Midbrain dopamine (da) neurons for engraftment

ABSTRACT

The present invention relates to the field of stem cell biology, in particular the lineage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC) in addition to nonembryonic human induced pluripotent stem cells (hiPSC), somatic stem cells, stem cells from patients with a disease, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and/or hiPSC into floor plate midbrain progenitor cells and then further into large populations of midbrain fate FOXA2 + LMX1A + TH +  dopamine (DA) neurons using novel culture conditions. The midbrain fate FOXA2 + LMX1A + TH +  dopamine (DA) neurons made using the methods of the present invention are further contemplated for various uses including, but not limited to, use in in vitro drug discovery assays, neurology research, and as a therapeutic to reverse disease of, or damage to, a lack of dopamine neurons in a patient. Further, compositions and methods are provided for differentiating midbrain fate FOXA2 + LMX1A + TH +  dopamine (DA) neurons from human pluripotent stem cells for use in disease modeling, in particular Parkinson&#39;s disease. Additionally, authentic DA neurons are enriched for markers, such as CD142, and A9 type neuronal cells.

This application is a Continuation of U.S. patent application Ser. No.16/353,546 filed Mar. 14, 2019, which is a Continuation of U.S. patentapplication Ser. No. 14/356,042 filed May 2, 2014 issued as U.S. Pat.No. 10,280,398, which is a U.S. National Stage Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2012/063339filed Nov. 2, 2012, which claims the benefit of priority to U.S.Provisional Patent Application No. 61/555,828 filed Nov. 4, 2011, thecontent of each of which is incorporated by reference in its entiretyherein, and to each of which priory is claimed.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant numbersNS047085 and NS052671 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of stem cell biology, inparticular the linage specific differentiation of pluripotent ormultipotent stem cells, which can include, but is not limited to, humanembryonic stem cells (hESC) in addition to nonembryonic human inducedpluripotent stem cells (hiPSC), somatic stem cells, stem cells frompatients with a disease, or any other cell capable of lineage specificdifferentiation. Specifically described are methods to direct thelineage specific differentiation of hESC and/or hiPSC into floor platemidbrain progenitor cells and then further into large populations ofmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons using novel cultureconditions. The midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons madeusing the methods of the present invention are further contemplated forvarious uses including, but not limited to, use in in vitro drugdiscovery assays, neurology research, and as a therapeutic to reversedisease of, or damage to, a lack of dopamine neurons in a patient.Further, compositions and methods are provided for differentiatingmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons from humanpluripotent stem cells for use in disease modeling, in particularParkinson's disease. Additionally, authentic DA neurons are enriched formarkers, such as CD142, and A9 type neuronal cells.

BACKGROUND OF THE INVENTION

Cell populations that retain the ability to differentiate into numerousspecialized cell types are useful for developing large numbers oflineage specific differentiated cell populations. These cell populationsthat retain a capability for further differentiation into specializedcells contain pluripotent cells. Pluripotent cells may be from embryonicand/or nonembryonic somatic stem cell origin.

These lineage specific differentiated cell populations are contemplatedto find use in cell replacement therapies for patients with diseasesresulting in a lose of function of a defined cell population. Inaddition to their direct therapeutic value, lineage specificdifferentiated cells are also valuable research tools for a variety ofpurposes including in vitro screening assays to identify, confirm, andtest for specification of function or for testing delivery oftherapeutic molecules to treat cell lineage specific disease.

Previously embryonic and somatic stem cells were used as therapeuticsand model systems for neurodegenerative diseases. Research andtechnological developments relating to directed differentiation ofembryonic and somatic stem cells has taken place in the field ofdiseases of the central nervous system (CNS), such as for Huntington's,Alzheimer's, Parkinson's, and multiple sclerosis. However the results ofthese studies showed little capability of these cells used in vivo toallow the patient to recover neuronal function and often resulted in thegrowth of unwanted tumors in the patients.

Therefore there is a need for compositions and methods to obtain cellpopulations capable of being used both in research and as a therapeuticfor treating diseases resulting in a loss of cells having a particularfunction.

SUMMARY OF THE INVENTION

The present invention relates to the field of stem cell biology, inparticular the linage specific differentiation of pluripotent ormultipotent stem cells, which can include, but is not limited to, humanembryonic stem cells (hESC) in addition to nonembryonic human inducedpluripotent stem cells (hiPSC), somatic stem cells, stem cells frompatients with a disease, or any other cell capable of lineage specificdifferentiation. Specifically described are methods to direct thelineage specific differentiation of hESC and/or hiPSC into floor platemidbrain progenitor cells and then further into large populations ofmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons using novel cultureconditions. The midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons madeusing the methods of the present invention are further contemplated forvarious uses including, but not limited to, use in in vitro drugdiscovery assays, neurology research, and as a therapeutic to reversedisease of, or damage to, a lack of dopamine neurons in a patient.Further, compositions and methods are provided for differentiatingmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons from humanpluripotent stem cells for use in disease modeling, in particularParkinson's disease. Additionally, authentic DA neurons are enriched formarkers, such as CD142, and A9 type neuronal cells.

A kit comprising a first signaling inhibitor, a second signalinginhibitor and a third signaling inhibitor, wherein said first inhibitoris capable of lowering transforming growth factor beta(TGFβ)/Activin-Nodal signaling, said second inhibitor is capable oflowering Small Mothers Against Decapentaplegic (SMAD) signaling and saidthird inhibitor is capable of lowering glycogen synthase kinase 3β(GSK3β) for activation of wingless (Wnt) signaling. In one embodiment,said first inhibitor is LDN-193189. In other embodiments, said firstinhibitor is selected from the group consisting of LDN-193189,derivatives thereof and mixtures thereof. In one embodiment, said secondinhibitor is SB431542. In other embodiments, said second inhibitor isselected from the group consisting of SB431542, derivatives thereof andmixtures thereof. In one embodiment, said third inhibitor is CHIR99021.In other embodiments, said third inhibitor is selected from the groupconsisting of CHIR99021, derivatives thereof and mixtures thereof. Inone embodiment, said the kit further comprises an activator of Sonichedgehog (SHH) signaling and an activator of fibroblast growth factor(FGF) 8 receptor family signaling. In one embodiment, said the kitfurther comprises brain-derived neurotrophic factor (BDNF), ascorbicacid (AA), glial cell line-derived neurotrophic factor, dibutyryl cAMPand transforming growth factor type ß3. In one embodiment, said the kitfurther comprise antibodies selected from the group consisting ofanti-tyrosine hydroxylase (TH), anti-forkhead box protein A2 (FOXA2),and anti-LIM homeobox transcription factor 1, alpha (LMX1A). In oneembodiment, said the kit further comprises a cell selected from thegroup consisting of a stem cell, embryonic stem cell, inducedpluripotent stem cell, and an engineered cell. In one embodiment, saidthe kit further comprises instructions for differentiating progenitorcells and midbrain fate FOXA2/LMX1A+ dopamine (DA) neurons. In oneembodiment, said the kit further comprises instructions for obtaining acell from a patient with Parkinson's disease (PD).

A composition, comprising, a cell population in contact with a firstsignaling inhibitor and a second signaling inhibitor, wherein greaterthan 40% of said cell population is positive for forkhead box protein A2(FOXA2), wherein said cell population was previously contacted by afirst signaling inhibitor, a third signaling inhibitor, and an activatorof Sonic hedgehog (SHH) signaling, wherein said first inhibitor iscapable of lowering transforming growth factor beta (TGFβ)/Activin-Nodalsignaling, said second inhibitor is capable of lowering glycogensynthase kinase 3β (GSK3β) signaling for activation of wingless (Wnt)signaling and said third inhibitor is capable of lowering Small MothersAgainst Decapentaplegic (SMAD) signaling. In one embodiment, said firstinhibitor is a small molecule selected from the group consisting ofLDN-193189, derivatives thereof and mixtures thereof. In one embodiment,said second inhibitor is selected from the group consisting of CHIR99021and derivatives thereof. In one embodiment, said third inhibitor isselected from the group consisting of SB431542, derivatives thereof andmixtures thereof. In one embodiment, said activator of Sonic hedgehog(SHH) signaling is selected from the group consisting of Sonic hedgehog(SHH) C25II and smoothened (SMO) receptor small molecule agonist,wherein said agonist is purmorphamine. In some embodiments, said cellpopulation was further previously contacted with Fibroblast growthfactor 8 (FGF8). In one embodiment, said majority of cells comprisingsaid cell population are forkhead box protein A2 (FOXA2)⁺LIM+ homeoboxtranscription factor 1+, alpha (LMX1A),⁺NGN2+ and DDC+ floor platemidbrain progenitor cells. In one embodiment, said cell population isselected from the group consisting of a rodent cells, primate cells andhuman cells. In one embodiment, said cells are derived from Parkinson'sdisease (PD) patient cells. In one embodiment, said cell population isat least 50% positive for forkhead box protein A2 (FOXA2). In oneembodiment, said cell population is at least 60% positive for forkheadbox protein A2. In one embodiment, said cell population is at least 70%positive for forkhead box protein A2. In one embodiment, said cellpopulation is at least 80% positive for forkhead box protein A2. In oneembodiment, said cell population is at least 90% positive for forkheadbox protein A2. In one embodiment, said cell population is at least 95%up to 100% positive for forkhead box protein A2.

A composition, comprising, an in vitro cell population wherein themajority of cells comprising said cell population are tyrosinehydroxylase (TH)⁺ forkhead box protein A2 (FOXA2)⁺ LIM homeoboxtranscription factor 1+, alpha (LMX1A)⁺ floor plate midbrain dopamine(DA) neurons. In one embodiment, said greater than 40% of said floorplate midbrain dopamine (DA) neurons are tyrosine hydroxylase positive(TH+). In one embodiment, said cell population is at least 50% tyrosinehydroxylase positive. In one embodiment, said cell population is atleast 60% tyrosine hydroxylase positive. In one embodiment, said cellpopulation is at least 70% tyrosine hydroxylase positive. In oneembodiment, said cell population is at least 80% tyrosine hydroxylasepositive. In one embodiment, said cell population is at least 90%tyrosine hydroxylase positive. In one embodiment, said cell populationis at least 95% up to 100% tyrosine hydroxylase positive. In someembodiments, said cell population comprises a majority of midbrain fateFOXA2/LMX1A+ dopamine (DA) neurons. In one embodiment, said floor platemidbrain dopamine (DA) neurons are positive for markers selected fromthe group consisting of nuclear receptor NURR1 (NR4A2), Neuron-specificclass III beta-tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3),achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), earlyB-cell factor 3 (EBF-3) and transthyretin (TTR). In one embodiment, saidmidbrain fate FOXA2/LMX1A+ dopamine (DA) neuron population is positivefor a molecule selected from the group consisting of DA,3,4-Dihydroxy-Phenylacetic Acid (DOPAC) and homovanillic acid (HVA). Inone embodiment, said marker is selected from the group consisting of aprotein and a nucleic acid. In some embodiments, said midbrain fateFOXA2/LMX1A+ dopamine (DA) neuron population is capable of engrafting invivo in a patient selected from the group consisting of a Parkinsondisease (PD) patient. In one embodiment, said midbrain fate FOXA2/LMX1A+dopamine (DA) neurons are capable of engrafting in vivo and providingdopamine (DA) neuronal function.

In some embodiments, the inventions provide a composition, comprising, acell population in contact with LDN-193189 and CHIR99021, whereingreater than 40% of said cell population is positive for forkhead boxprotein A2 (FOXA2), and wherein said cell population was previouslycontacted by LDN-193189, SB431542, an activator of Sonic hedgehog (SHH)signaling and CHIR99021. In one embodiment, said activator of Sonichedgehog (SHH) signaling is selected from the group consisting of Sonichedgehog (SHH) C25II and purmorphamine. In one embodiment, said greaterthan 10% of said cell population is double positive for forkhead boxprotein A2 (FOXA2) and LIM homeobox transcription factor 1, alpha(LMX1A). In one embodiment, said majority of said cell population is apopulation of floor plate midbrain progenitor cells. In one embodiment,said cell population was previously contacted with fibroblast growthfactor 8 (FGF8). In one embodiment, said cell population is selectedfrom the group consisting of rodent cells, primate cells and humancells. In one embodiment, said human cells are cells from a patient witha neurological symptom of Parkinson's disease (PD). In one embodiment,said cell population is derived from an induced pluripotent stem cell(iPSC). In one embodiment, greater than 10% of said cell population isselected from the group consisting of double positive for forkhead boxprotein A2 (FOXA2)/LIM homeobox transcription factor 1, alpha (LMX1A)and double positive for forkhead box protein A2 (FOXA2)/orthodenticlehomeobox 2 (OTX2).

In one embodiment, the inventions provide a method for inducing directeddifferentiation of cells into a population of floor plate midbrainprogenitor cells, comprising, a) providing: i) a cell population,wherein said cell population is selected from the group consisting of anonembryonic stem cell, an embryonic stem cell, an induced nonembryonicpluripotent cell and an engineered pluripotent cell; and ii) a firstsignaling inhibitor, a second signaling inhibitor, an activator of Sonichedgehog (SHH) signaling and a third signaling inhibitor, wherein saidfirst inhibitor is capable of lowering transforming growth factor beta(TGFβ)/Activin-Nodal signaling, said second inhibitor is capable oflowering Small Mothers Against Decapentaplegic (SMAD) signaling and saidthird inhibitor is capable of lowering glycogen synthase kinase 3β(GSK3β) signaling for activation of wingless (Wnt) signaling; b)contacting said cell population with said first and said secondinhibitor, c) after contacting said cell population with said first andsaid second inhibitor further contacting said cells with said activatorof Sonic hedgehog (SHH) signaling under conditions for differentiating apopulation of floor plate midbrain progenitor cells; and) aftercontacting said cell population with said activator of Sonic hedgehog(SHH) signaling further contacting said cells with said third inhibitorfor differentiating said cell population into a population of floorplate midbrain progenitor cells. In one embodiment, said contact withsaid first and said second inhibitor is under conditions capable ofresulting in said differentiated population of floor plate midbrainprogenitor cells. In one embodiment, said contact with said first andsaid second inhibitor is within 1 hour of plating cells in vitro. In oneembodiment, said contact with said first and said second inhibitor iswithin 48 hours of plating cells in vitro. In one embodiment, saidcontact with said first and said second inhibitor is within 62 hours ofplating cells in vitro. In one embodiment, said contact of said cellswith said activator of Sonic hedgehog (SHH) signaling is underconditions capable of resulting in said differentiated population offloor plate midbrain progenitor cells. In one embodiment, said contactof said cells with said activator of Sonic hedgehog (SHH) signaling isat least 24 hours up to 36 hours after contacting said cell populationwith said first and said second inhibitor. In one embodiment, saidcontact of said cells with said activator of Sonic hedgehog (SHH)signaling is up to 144 hours. In one embodiment, said contact of saidcells with said third inhibitor is under conditions capable of resultingin said differentiated population of floor plate midbrain progenitorcells. In one embodiment, said contact of said cells with said thirdinhibitor is at least 24 hours up to 36 hours after contacting said cellpopulation with said activator of Sonic hedgehog (SHH) signaling. In oneembodiment, said contact of said cells with said third inhibitor is upto 192 hours. In one embodiment, said cell population is differentiatedinto said floor plate midbrain progenitor cells by at least day 11 aftercontacting said cells with said first and said second inhibitor. In oneembodiment, said first inhibitor is SB431542. In one embodiment, saidsecond inhibitor is LDN-193189. In one embodiment, said third inhibitoris CHIR99021. In one embodiment, said activator of Sonic hedgehog (SHH)signaling is selected from the group consisting of Sonic hedgehog (SHH)C25II and purmorphamine. In one embodiment, said method further providesFibroblast growth factor 8 (FGF8) and contacting said cell populationwith said FGF8 under conditions capable of resulting in saiddifferentiated population of floor plate midbrain progenitor cells. Inone embodiment, said contact of said cells with said FGF8 is at least 24up to 36 hours after contacting said cell population with said first andsaid second inhibitor. In one embodiment, said contact of said cellswith said FGF8 is up to 144 hours. In one embodiment, said floor platemidbrain progenitor cell population comprises greater than 40% forkheadbox protein A2 (FOXA2)⁺ cells. In one embodiment, said floor platemidbrain progenitor cell population comprises greater than 40% forkheadbox protein A2 (FOXA2)⁺LIM homeobox transcription factor 1, alpha(LMX1A)⁺ cells. In one embodiment, said method further comprises step e)contacting said population of floor plate midbrain progenitor cells withneuronal maturation medium, said medium comprising N2 medium,brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cellline-derived neurotrophic factor, dibutyryl cAMP (dbcAMP) andtransforming growth factor type ß3 for differentiation of floor platemidbrain progenitor cells into floor plate midbrain dopamine (DA)neurons. In one embodiment, said method further comprises step e)contacting said population of floor plate midbrain progenitor cells withneuronal maturation medium with B27 supplement for differentiation offloor plate midbrain progenitor cells into floor plate midbrain dopamine(DA) neurons. In one embodiment, said cells contacted with neurobasalmedium with B27 supplement are contacted with brain-derived neurotrophicfactor (BDNF), ascorbic acid (AA), glial cell line-derived neurotrophicfactor, dibutyryl cAMP (dbcAMP) and transforming growth factor type ß3for differentiation of floor plate midbrain progenitor cells into floorplate midbrain dopamine (DA) neurons. In one embodiment, said floorplate midbrain dopamine (DA) neurons are forkhead box protein A2(FOXA2)⁺LIM homeobox transcription factor 1, alpha (LMX1A)⁺, Nuclearreceptor related 1 protein (NURR1)⁺ and tyrosine hydroxylase (TH)⁺. Inone embodiment, greater than 40% of said floor plate midbrain dopamine(DA) neurons are tyrosine hydroxylase (TH)⁺. In one embodiment, saidpopulation of floor plate midbrain dopamine (DA) neurons aredifferentiated by at least day 25 after contacting said cell populationwith said first and said second inhibitor. In one embodiment, said floorplate midbrain dopamine (DA) neurons are positive for markers thatidentify molecules. In one embodiment, said markers are selected fromthe group consisting of tyrosine hydroxylase (TH), forkhead box proteinA2 (FOXA2), LEVI homeobox transcription factor 1, dompamine,3,4-Dihydroxy-Phenylacetic Acid (DOPAC) and homovanillic acid (HVA),alpha, nuclear receptor NURR1 (NR4A2), Neuron-specific class IIIbeta-tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3),achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), earlyB-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopaminetransporter (DAT), and G-protein coupled, and inwardly rectifyingpotassium channel (Kir3.2/GIRK2). In one embodiment, said molecule isselected from the group consisting of a protein and a nucleic acid. Inone embodiment, said molecule is identified using a marker selected fromthe group consisting of an antibody, a PCR primer, a nucleic acidsequence and an enzyme assay. In one embodiment, said floor platemidbrain dopamine (DA) neurons are capable of engrafting in vivo in apatient with Parkinson disease (PD) for providing dopamine (DA) neuronalfunction. In one embodiment, said method further comprises, providing, apatient in need of dopamine producing neurons, wherein said patientshows at least one neurological symptom, and the step of transplantingfloor plate midbrain dopamine (DA) neurons into said patient forproviding dopamine (DA) neuronal function. In one embodiment, saidneurological symptoms are selected from the group consisting of tremor,bradykinesia (extreme slowness of movement), flexed posture, posturalinstability, and rigidity. In one embodiment, said patient shows areduction of said neurological symptom. In one embodiment, said cell isselected from a rodent cell, a primate cell and a human cell. In oneembodiment, said human cells are cells from a patient with aneurological symptom of Parkinson's disease (PD).

In one embodiment, the inventions provide a method of engrafting in vivofor therapeutic treatment, comprising, a) providing: i) a population offloor plate midbrain dopamine (DA) neurons wherein greater than 40% ofsaid population expresses tyrosine hydroxylase (TH); and ii) a subject,wherein said subject shows at least one neurological symptom; and b)transplanting said floor plate midbrain dopamine (DA) neurons into saidsubject under conditions for allowing in vivo engraftment for providingdopamine (DA) neuronal function. In one embodiment, said neurologicalsymptoms are selected from the group consisting of tremor, bradykinesia(extreme slowness of movement), flexed posture, postural instability andrigidity. In one embodiment, said subject shows reduction of saidneurological symptom. In one embodiment, said population of floor platemidbrain dopamine (DA) neurons are derived from a population of floorplate midbrain progenitor cells treated according to methods of thepresent inventions. In one embodiment, said population of floor platemidbrain dopamine (DA) neurons are derived from a population of floorplate midbrain progenitor cells treated according to a method furthercomprising a step of contacting said population of floor plate midbrainprogenitor cells with neuronal maturation medium, said medium comprisingN2 medium, brain-derived neurotrophic factor (BDNF), ascorbic acid (AA),glial cell line-derived neurotrophic factor, dibutyryl cAMP andtransforming growth factor type ß3 for differentiation of floor platemidbrain progenitor cells into floor plate midbrain dopamine (DA)neurons. In one embodiment, said population of floor plate midbrainprogenitor cells are derived from a cell population treated according toa method of the present inventions. In one embodiment, said populationof floor plate midbrain progenitor cells are derived from a cellpopulation treated according to a method for inducing directeddifferentiation of cells into a population of floor plate midbrainprogenitor cells, comprising, a) providing: i) a cell population,wherein said cell population is selected from the group consisting of anonembryonic stem cell, an embryonic stem cell, an induced nonembryonicpluripotent cell and an engineered pluripotent cell; and ii) a firstsignaling inhibitor, a second signaling inhibitor, an activator of Sonichedgehog (SHH) signaling and a third signaling inhibitor, wherein saidfirst inhibitor is capable of lowering transforming growth factor beta(TGFβ)/Activin-Nodal signaling, said second inhibitor is capable oflowering Small Mothers Against Decapentaplegic (SMAD) signaling and saidthird inhibitor is capable of lowering glycogen synthase kinase 3β(GSK3β) signaling for activation of wingless (Wnt) signaling; b)contacting said cell population with said first and said secondinhibitor, c) after contacting said cell population with said first andsaid second inhibitor further contacting said cells with said activatorof Sonic hedgehog (SHH) signaling under conditions for differentiating apopulation of floor plate midbrain progenitor cells; and d) aftercontacting said cell population with said activator of Sonic hedgehog(SHH) signaling further contacting said cells with said third inhibitorfor differentiating said cell population into a population of floorplate midbrain progenitor cells. In one embodiment, said population offloor plate midbrain dopamine (DA) neurons are derived from a cellpopulation selected from the group consisting of animals, primates andhumans. In one embodiment, said human cells are cells from a patientwith a symptom of Parkinson's disease (PD).

In one embodiment, the inventions provide a composition, comprising, acell population in contact with LDN-193189 and CHIR99021, whereingreater than 40% of said cell population is positive for forkhead boxprotein A2 (FOXA2), and wherein said cell population was previouslycontacted by LDN-193189, SB431542, an activator of Sonic hedgehog (SHH)signaling and CHIR99021, wherein said activator of Sonic hedgehog (SHH)signaling is selected from the group consisting of Sonic hedgehog (SHH)C25II and purmorphamine. In one embodiment, said cell population is atleast 50% positive for forkhead box protein A2 (FOXA2). In oneembodiment, said cell population is at least 60% positive for forkheadbox protein A2. In one embodiment, said cell population is at least 70%positive for forkhead box protein A2. In one embodiment, said cellpopulation is at least 80% positive for forkhead box protein A2. In oneembodiment, said cell population is at least 90% positive for forkheadbox protein A2. In one embodiment, said cell population is at least 95%up to 100% positive for forkhead box protein A2. In one embodiment,greater than 10% of said cell population is selected from the groupconsisting of double positive for forkhead box protein A2 (FOXA2)/LIMhomeobox transcription factor 1, alpha (LMX1A) and double positive forforkhead box protein A2 (FOXA2)/orthodenticle homeobox 2 (OTX2). In oneembodiment, said cell population is at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, at least 95% up to 100% positive. In one embodiment, atleast 20% of said cell population is positive for a marker selected fromthe group consisting of Nurr1+, CD142, DCSM1, CD63 and CD99. In someembodiments, said cell population is at least 30%, 40%, 50%, 60%, 70%,80%, 90%, at least 95% up to 100% positive. In some embodiments, saidcell population is at least 50%, 60%, 70%, 80%, 90%, at least 95% up to100% positive. In one embodiment, said cell population is selected fromthe group consisting of rodent cells, primate cells, human cells andhuman cells from a patient with a neurological symptom of Parkinson'sdisease (PD).

In one embodiment, the inventions provide a method for inducing directeddifferentiation of cells into a population of floor plate midbrainprogenitor cells, comprising, a) providing: i) a cell population,wherein said cell population is selected from the group consisting of anonembryonic stem cell, an embryonic stem cell, an induced nonembryonicpluripotent cell and an engineered pluripotent cell; and ii) a firstsignaling inhibitor, a second signaling inhibitor, an activator of Sonichedgehog (SHH) signaling and a third signaling inhibitor, wherein saidfirst inhibitor is SB431542, said second inhibitor is LDN-193189, saidactivator of Sonic hedgehog (SHH) signaling is selected from the groupconsisting of Sonic hedgehog (SHH) C25II and a purmorphaminc, and saidthird inhibitor is CHIR99021; b) contacting said cell population withsaid first and said second inhibitor, wherein said contact with saidfirst and said second inhibitor is under conditions capable of resultingin said differentiated population of floor plate midbrain progenitorcells such that said contact with said first and said second inhibitoris within 48 hours of plating cells in vitro, c) after contacting saidcell population with said first and said second inhibitor furthercontacting said cells with said activator of Sonic hedgehog (SHH)signaling under conditions for differentiating a population of floorplate midbrain progenitor cells; and d) after contacting said cellpopulation with said activator of Sonic hedgehog (SHH) signaling furthercontacting said cells with said third inhibitor for differentiating saidcell population into a population of floor plate midbrain progenitorcells. In one embodiment, contact of said cells with said activator ofSonic hedgehog (SHH) signaling is under conditions capable of resultingin said differentiated population of floor plate midbrain progenitorcells such that said contact of said cells with said activator of Sonichedgehog (SHH) signaling is at least 24 hours and up to 36 hours aftercontacting said cell population with said first and said secondinhibitor. In one embodiment, contact of said cells with said thirdinhibitor is under conditions capable of resulting in saiddifferentiated population of floor plate midbrain progenitor cells suchthat said contact of said cells with said third inhibitor is at least 24hours and up to 36 hours after contacting said cell population with saidactivator of Sonic hedgehog (SHH) signaling. In one embodiment, floorplate midbrain progenitor cell population comprises greater than 40%forkhead box protein A2 (FOXA2)⁺LIM homeobox transcription factor 1+,alpha (LMX1A)⁺ cells. In one embodiment, the method further comprisesstep e) contacting said population of floor plate midbrain progenitorcells with neuronal maturation medium, said medium comprising N2 medium,brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cellline-derived neurotrophic factor, dibutyryl cAMP and transforming growthfactor type ß3 for differentiation of floor plate midbrain progenitorcells into floor plate midbrain dopamine (DA) neurons. In oneembodiment, said floor plate midbrain dopamine (DA) neurons are positivefor sets of markers selected from the group consisting of forkhead boxprotein A2 (FOXA2)^(/)LIM homeobox transcription factor 1 alpha(LMX1A)/tyrosine hydroxylase (TH); forkhead box protein A2/LIM homeoboxtranscription factor 1 alpha/tyrosine hydroxylase/CD142; forkhead boxprotein A2/LIM homeobox transcription factor 1 alpha/tyrosinehydroxylase/Nuclear receptor related 1 protein (NURR1); forkhead boxprotein A2/LIM homeobox transcription factor 1 alpha/tyrosinehydroxylase/CD142/Nuclear receptor related 1 protein and tyrosinehydroxylase/α-synuclein. In one embodiment, said method furthercomprises step f) sorting said floor plate midbrain dopamine (DA)neurons for CD142 expression into a population of cells at least 80%positive for CD142. In one embodiment, said floor plate midbraindopamine (DA) neurons are positive for a marker that identifies amolecule selected from the group consisting of tyrosine hydroxylase(TH), forkhead box protein A2 (FOXA2), LIM homeobox transcription factor1, dompamine, 3,4-Dihydroxy-Phenylacetic Acid (DOPAC) and homovanillicacid (HVA), alpha, nuclear receptor NURR1 (NR4A2), Neuron-specific classIII beta-tubulin (Tuj1), TTF3, paired-like homeodomain 3 (PITX3),achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), earlyB-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopaminetransporter (DAT), and G-protein coupled, inwardly rectifying potassiumchannel (Kir3.2/GIRK2), CD142, DCSM1, CD63 and CD99. In one embodiment,the method further comprises, provides, a patient in need of dopamineproducing neurons, and a step after e) treating said patient bytransplanting said floor plate midbrain dopamine (DA) neurons forproviding dopamine (DA) neuronal function. In one embodiment, saidpatient comprises at least one neurological symptom selected from thegroup consisting of tremor, bradykinesia (extreme slowness of movement),flexed posture, postural instability, and rigidity. In one embodiment,said patient is observed to have at least one neurological symptomselected from the group consisting of tremor, bradykinesia (extremeslowness of movement), flexed posture, postural instability, andrigidity. In one embodiment, said patient shows a reduction of at leastone of said neurological symptom.

In one embodiment, the inventions provide a method of engrafting in vivofor therapeutic treatment, comprising, a) providing: i) a population offloor plate midbrain dopamine (DA) neurons wherein greater than 40% ofsaid population expresses tyrosine hydroxylase (TH); and ii) a subject,wherein said subject shows at least one neurological symptom, whereinsaid neurological symptoms are selected from the group consisting oftremor, bradykinesia (extreme slowness of movement), flexed posture,postural instability and rigidity; and b) transplanting said floor platemidbrain dopamine (DA) neurons into said subject under conditions forallowing in vivo engraftment for providing dopamine (DA) neuronalfunction. In one embodiment, said subject shows reduction of at leastone of said neurological symptom. In one embodiment, said population offloor plate midbrain dopamine (DA) neurons are derived from a populationof cells further comprising a step of sorting said floor plate midbraindopamine (DA) neurons for CD142 expression into a population of cells atleast 80% positive for CD142. In one embodiment, said population offloor plate midbrain dopamine (DA) neurons are derived from a populationof floor plate midbrain progenitor cells after a step of sorting saidfloor plate midbrain dopamine (DA) neurons for CD142 expression into apopulation of cells at least 80% positive for CD142. In one embodiment,said population of floor plate midbrain dopamine (DA) neurons arederived from a cell population selected from the group consisting ofanimals, primates, humans and a patient with a symptom of Parkinson'sdisease (PD). In one embodiment, said population of floor plate midbraindopamine (DA) neurons are derived from a cell population isolated fromthe group consisting of animals, primates, humans and a patient with asymptom of Parkinson's disease (PD).

Definitions

As used herein, the term “disease modeling” refers to the process ofusing an experimental organism or in vitro cell cultures to mimicspecific signs or symptoms observed in humans as a result of a disorder.In one embodiment, pluripotent stem cells derived from an animal modelwith a genetic mutation resulting in a neurological disorder, such asParkinson's disease (PD), can be grown and differentiated into neuralcells for identifying new characteristics of neurons related to PD. Inone embodiment, human pluripotent stem cells derived from a person witha genetic mutation resulting in a neurological disorder, such asParkinson's disease (PD) can be grown and differentiated into neuralcells harboring a similar defect observed within the person.

As used herein, term “parkinsonism” refers to a group of diseases thatare all linked to an insufficiency of dopamine in the basal gangliawhich is a part of the brain that controls movement. Symptoms includetremor, bradykinesia (extreme slowness of movement), flexed posture,postural instability, and rigidity. A diagnosis of parkinsonism requiresthe presence of at least two of these symptoms, one of which must betremor or bradykinesia. The most common form of parkinsonism isidiopathic, or classic, Parkinson's disease (PD), but for a significantminority of diagnoses, about 15 percent of the total, one of theParkinson's plus syndromes (PPS) may be present. These syndromes alsoknown as atypical parkinsonism, include corticobasal degeneration, Lewybody dementia, multiple systematrophy, and progressive supranuclearpalsy. In general, Parkinson's disease involves the malfunction anddeath of vital nerve cells in the brain primarily in an area of thebrain called the substantia nigra. Many of these vital nerve cells makedopamine, that as these neurons die off, the amount of dopamineresulting from differentiation in the brain decreases, leaving a personunable to control movement normally. The intestines also have dopaminecells that degenerate in Parkinson's disease patients, and this may bean important causative factor in the gastrointestinal symptoms that arepart of the disease. A group of symptoms that an individual experiencesvaries from person to person. Primary motor signs of Parkinson's diseaseinclude the following: tremor of the hands, arms, legs, jaw and face,bradykinesia or slowness of movement, rigidity or stiffness of the limbsand trunk and postural instability or impaired balance and coordination.

As used herein, the term “subject” refers to a mammal (human and animal,i.e. non-human animals) that is to be the recipient of a particulartreatment including any type of control. Typically, the terms “subject”and “patient” are used interchangeably herein in reference to a humansubject.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, term “dopamine” refers to a chemical made by dopamineneurons that sends messages to the part of the brain containing neuronsthat control movement and coordination.

As used herein, the term “LSB” refers to a combination of two compoundsLDN-193189 and SB431542 capable of lowering or blocking signalingconsisting of transforming growth factor beta (TGFβ)/Activin-Nodalsignaling and Small Mothers Against Decapentaplegic (SMAD) signaling ina cell.

As used herein, the term “SB431542” refers to a molecule capable oflowering or blocking transforming growth factor beta(TGFβ)/Activin-Nodal signaling with a number CAS 301836-41-9, amolecular formula of C₂₂H₁₈N₄O₃, and a name of4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide,for example, see structure below:

In one exemplary, SB431542 is Stemolecule™ SB431542, Stemgent, Inc.Cambridge, Mass., United States.

As used herein, the term “LDN-193189” refers to a small moleculeDM-3189, IUPAC name4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline,with a chemical formula of C₂₅H₂₂N₆. LDN-193189 is capable offunctioning as a SMAD signaling inhibitor. LDN-193189 is also a highlypotent small-molecule inhibitor of ALK2, ALK3, and ALK6, proteintyrosine kinases (PTK), inhibiting signaling of members of the ALK1 andALK3 families of type I TGFβ receptors, resulting in the inhibition ofthe transmission of multiple biological signals, including the bonemorphogenetic proteins (BMP) BMP2, BMP4, BMP6, BMP7, and Activincytokine signals and subsequently SMAD phosphorylation of Smad1, Smad5,and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008)Bioorg. Med. Chem. Lett. 18: 4388-4392, herein incorporated byreference).

In one exemplary embodiment, LDN-193189 is Stemolecule™ LDN-193189,Stemgent, Inc. Cambridge, Mass., United States.

As used herein, the term “glycogen synthase kinase 3β inhibitor” or“GSK3β inhibitor” refers to a compound that inhibits a glycogen synthasekinase 3β enzyme, for example, see, Doble, et al., J Cell Sci. 2003;116:1175-1186, herein incorporated by reference. For the purposes of thepresent inventions, a GSK3P inhibitor is capable of activating a WNTsignaling pathway, see, for example, Cadigan, et al., J Cell Sci. 2006;119:395-402; Kikuchi, et al., Cell Signaling. 2007; 19:659-671, hereinincorporated by reference.

As used herein, the term “CHIR99021” or “CHIR” or “aminopyrimidine” or“3-[3-(2-Carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone”refers to IUPAC name6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile.CHIR99021 is one example of a small-molecule chemical inhibitor ofglycogen synthase kinase 3P (GSK3P) that activates a WNT signalingpathway, and is highly selective, showing nearly thousand foldselectivity against a panel of related and unrelated kinases, with anIC₅₀=6.7 nM against human GSK3p and nanomolar IC₅₀ values against rodentGSK3P homologs.

In one exemplary embodiment, CHIR99021 is Stemolecule™ CHIR99021,Stemgent, Inc. Cambridge, Mass., United States.

As used herein, the term “purmorphamine” refers to a purine derivative,such as CAS Number: 483367-10-8, for one example see structure below,that activates the Hedgehog pathway including by targeting Smoothened.

In one exemplary embodiment, purmorphamine is Stemolecule™Purmorphamine, Stemgent, Inc. Cambridge, Mass., United States.

As used herein, the term “signaling” in reference to a “signaltransduction protein” refers to proteins that are activated or otherwiseaffected by ligand binding to a membrane receptor protein or some otherstimulus. Examples of signal transduction protein include a SMAD, a WNTcomplex protein, in another embodiment a WNT complex protein includingbeta-catenin, Sonic hedgehog (SHH), NOTCH, transforming growth factorbeta (TGFβ), Activin, Nodal, glycogen synthase kinase 3β (GSK3β)proteins and the like. For many cell surface receptors or internalreceptor proteins, ligand-receptor interactions are not directly linkedto the cell's response. The ligand activated receptor must firstinteract with other proteins inside the cell before the ultimatephysiological effect of the ligand on the cell's behavior is produced.Often, the behavior of a chain of several interacting cell proteins isaltered following receptor activation or inhibition. The entire set ofcell changes induced by receptor activation is called a signaltransduction mechanism or signaling pathway.

As used herein, the term “LSB/S/F8/CHIR” or “LSB/SHH/FGF8/CHIR” refersto contacting cells with LDN-193189 and SB431542 (i.e. LSB) in additionto S, Sonic Hedgehog activator, F8, FGF8, and CHIR of the presentinventions. In contrast to “LSB/S/F8” or “SHH/FGF8” or “SHH/FGF” whichrefers to contacting cells with LDN-193189 and SB431542 (i.e. LSB) inaddition to S, Sonic Hedgehog activator, F8, FGF8 but without CHIR as inpreviously published methods. In similar abbreviations, LDN/SB” refersto contacting cells with LDN, LDN-193189 and SB, SB431542.

As used herein, the term “inhibit” or “block” means a reduction in thelevel of activity of a particular signaling pathway of a cell upontreatment with a compound (i.e. an inhibitor) compared to the activityof said signaling pathway of a cell that is left untreated with suchcompound or treated with a control.

As used herein, the term ““activate” means an increase in the level ofactivity of a particular signaling pathway of a cell upon treatment witha compound (i.e. an activator) compared to the activity of saidsignaling pathway of a cell that is left untreated with such compound ortreated with a control. Any level of inhibition or activation of aparticular signaling pathway is considered an embodiment of theinvention if such inhibition or activation results in the directeddifferentiation of a stem cell.

As used herein, the term “Sma Mothers Against Decapentaplegic” or “SmallMothers Against Decapentaplegic” or “SMAD” refers to a signalingmolecule.

As used herein, the term “WNT” or “wingless” in reference to a ligandrefers to a group of secreted proteins (i.e. Intl (integration 1) inhumans) capable of interacting with a WNT receptor, such as a receptorin the Frizzled and LRPDerailed/RYK receptor family.

As used herein, the term “WNT” or “wingless” in reference to a signalingpathway refers to a signal pathway composed of Wnt family ligands andWnt family receptors, such as Frizzled and LRPDerailedJRYK receptors,mediated with or without β-catenin. For the purposes described herein, apreferred WNT signaling pathway includes mediation by β-catenin, i.e.WNT/β-catenin.

As used herein, “canonical pathway” or “classical activation” inreference to WNT refers to one of the multiple Wnt downstream signalpathways, for example, in the canonical pathway a major effect of Wntligand binding to its receptor is the stabilization of cytoplasmicbeta-catenin through inhibition of the bea-catenin degradation complex.Others Wnt pathways are non-canonical.

As one example, the small molecule CHIR affects a canonical Wntsignaling downstream pathway.

As used herein, the term “Sonic hedgehog (SHH or Shh)” refers to aprotein that is one of at least three proteins in the mammaliansignaling pathway family called hedgehog, another is desert hedgehog(DHH) wile a third is Indian hedgehog (IHH). Shh interacts with at leasttwo transmembrane proteins by interacting with transmembrane moleculesPatched (PTC) and Smoothened (SMO). Shh typically binds to PCT whichthen allows the activation of SMO as a signal transducer. In the absenceof SHH, PTC typically inhibits SMO, which in turn activates atranscriptional repressor so transcription of certain genes does notoccur. When Shh is present and binds to PTC, PTC cannot interfere withthe functioning of SMO. With SMO uninhibited, certain proteins are ableto enter the nucleus and act as transcription factors allowing certaingenes to be activated (see, Gilbert, 2000 Developmental Biology(Sunderland, Mass.: Sinauer Associates, Inc., Publishers).

As used herein, the term “activator” or “activating” refers to smallmolecules, peptides, proteins and compounds for activating moleculesresulting in directed differentiation of cells of the presentinventions. Exemplary activators include but are not limited to: CHIR,Sonic hedgehog (SHH) C25II, a small molecule Smoothened agonistpurmorphamine, fibroblast growth factor (FGF), etc.

As used herein, the term “activator of Sonic hedgehog (SHH) signaling”refers to any molecule or compound that activates a SHH signalingpathway including a molecule or compound that binds to PCT or aSmoothened agonist and the like. Examples of such compounds are aprotein Sonic hedgehog (SHH) C25II and a small molecule Smoothenedagonist purmorphamine.

As used herein, the term “Sonic hedgehog (SHH) C25II” refers to arecombinant N-Terminal fragment of a full-length murine sonic hedgehogprotein capable of binding to the SHH receptor for activating SHH, oneexample is R and D Systems catalog number: 464-SH-025/CF.

As used herein, the term “signals” refer to internal and externalfactors that control changes in cell structure and function. They arechemical or physical in nature.

As used herein, the term “ligand” refers to molecules and proteins thatbind to receptors (R), examples include but are not limited totransforming growth factor-beta, activins, nodal, bone morphogenicproteins (BMPs), etc.

As used herein, the term “inhibitor” or “signaling inhibitor” is inreference to inhibiting a signaling molecule or a signaling molecule'spathway, such as an inhibitor of SMAD signaling, inhibitor of glycogensynthase kinase 3β (GSK3β) refers to a compound or molecule (e.g., smallmolecule, peptide, peptidomimetic, natural compound, protein, siRNA,anti sense nucleic acid, aptamer, or antibody) that interferes with(i.e. reduces or suppresses or eliminates or blocks) the signalingfunction of the molecule or pathway. In other words, an inhibitor is anycompound or molecule that changes any activity of a named protein(signaling molecule, any molecule involved with the named signalingmolecule, a named associated molecule, such as a glycogen synthasekinase 3β (GSK3β)) (e.g., including, but not limited to, the signalingmolecules described herein), for one example, via directly contactingSMAD signaling, contacting SMAD mRNA, causing conformational changes ofSMAD, decreasing SMAD protein levels, or interfering with SMADinteractions with signaling partners (e.g., including those describedherein), and affecting the expression of SMAD target genes (e.g. thosedescribed herein). Inhibitors also include molecules that indirectlyregulate SMAD biological activity by intercepting upstream signalingmolecules. Thus in one embodiment, an inhibitor of the presentinventions induces (changes) or alters differentiation from a default toa non-default cell type, for example, one of the methods of the presentinventions comprising LDN/SB, CHIR and a SHH activator (which mayinhibit glycogen synthase kinase 3β) differentiated progenitor cellsinto non-default neural progenitor cells. In a preferred embodiment, aninhibitor of the present inventions “alters” or “lowers” or “blocks”default signaling in order to direct cellular differentiation towards anondefault cell type, such as described herein for differentiating floorplate midbrain progenitor cells and midbrain fate FOXA2/LMX1A+ dopamine(DA) neurons of the present inventions. Thus, an inhibitor of thepresent inventions is a natural compound or small molecule which changessignal molecule activity in a manner that contributes to differentiationof a starting cell population (day 0) into floor plate midbrainprogenitor cells. When progenitor cells are contacted with inhibitorsthese small molecules may contribute to further differentiation intomidbrain fate FOXA2/LMX1A+ dopamine (DA) neurons of the presentinventions Inhibitors are described in terms of competitive inhibition(binds to the active site in a manner as to exclude or reduce thebinding of another known binding compound) and allosteric inhibition(binds to a protein in a manner to change the protein conformation in amanner which interferes with binding of a compound to that protein'sactive site) in addition to inhibition induced by binding to andaffecting a molecule upstream from the named signaling molecule that inturn causes inhibition of the named molecule. In some cases, aninhibitor is referred to as a “direct inhibitor” which refers toinhibiting a signaling target or a signaling target pathway by actuallycontacting the signaling target; for example, a direct inhibitor of agamma secretase is a DAPT molecule that binds to the gamma secretaseprotein.

As used herein, the term “derivative” refers to a chemical compound witha similar core structure.

As used herein, the term “floor plate midbrain progenitor cell” inreference to an in vivo cell located in a midbrain, including duringembryonic development of midbrain neurons, refers to a cell that maydifferentiate into a dopamine producing cell. In some embodiments, a“floor plate midbrain progenitor cell” refers to a cell in culture thatis used to artificially produce a cultured cell in vitro that expressesoverlapping or identical sets of markers when compared to markersexpressed by in vivo cells, i.e. co-expression of the floor plate markerFOXA2 and the roof plate marker LMX1A, OTX2, NGN2, and DDC, such as incultured cells of the present inventions around day 11 after initiationof directed differentiation as described herein. Preferably, a floorplate midbrain progenitor cell is “FOXA2+LMX1A+” or “FOXA2/LMX1A+”. Insome embodiments, low numbers of cells in a differentiated progenitorpopulation are FOXA2/LMX1A/TH+.

As used herein, the term “floor-plate derived DA neurons” or “authenticmidbrain DA neurons” or “midbrain fate FOXA2+LMX1A+ dopamine (DA)neurons” or “floor plate midbrain dopamine (DA) neuron” or “engraftablemidbrain DA neuron” or “mDA neuron” or “FOXA2+LMX1A+TH+” or“FOXA2/LMX1A/TH” or “FOXA2+LMX1A+NURR1+TH+” or “FOXA2/LMX1A/NURR1/TH”refers to an engraftable midbrain DA neuron population obtained bymethods described herein, typically around or by day 25 after initiatingdirected differentiation. In a preferred embodiment, “authentic midbrainDA neurons” are FOXA2+/LMX1A+/NURR1+/TH+. These neurons were labeled“engraftable” after transplantation experiments in mice and primatesshowing the capability of these neurons to reverse Parkinson-likeneurological conditions with less interference from neural overgrowthand teratoma formation. The midbrain fate FOXA2/LMX1A+ dopamine (DA)neurons of the present inventions were maintained in vitro for severalmonths while retaining engrafting capability.

As used herein, cells used for obtaining floor plate midbrain progenitorcells and midbrain fate FOXA2/LMX1A+ dopamine (DA) neurons are obtainedfrom a variety of sources including embryonic and nonembryonic sources,for example, hESCs and nonembryonic hiPSCs, somatic stem cells, diseasestem cells, i.e. isolated pluripotent cells and engineered derived stemcells isolated from Parkinson disease patients, cancer stem cells, humanor mammalian pluripotent cells, etc.

As used herein, the term “stem cell” refers to a cell with the abilityto divide for indefinite periods in culture and to give rise tospecialized cells. A stem cell may be obtained from animals andpatients, including humans; for example, a human stem cell refers to astem cell that is human. A stem cell may be obtained from a variety ofsources including embryonic and nonembryonic, such as umbilical cordcells, cells from children and cells from adults. For the purposes ofthe present inventions, adult stem cells in general refer to cells thatwere not originally obtained from a fetus, in other words, cells frombabies, cast off umbilical cords, cast off placental cells, cells fromchildren, cells from adults, etc.

As used herein, the term “umbilical cord blood stem cells” refer to stemcells collected from an umbilical cord at birth that have the capabilityto at least produce all of the blood cells in the body (hematopoietic).

As used herein, the term “somatic (adult) stem cell” refers to arelatively rare undifferentiated cell found in many organs anddifferentiated tissues with a limited capacity for both self renewal (inthe laboratory) and differentiation. Such cells vary in theirdifferentiation capacity, but it is usually limited to cell types in theorgan of origin.

As used herein, the term “somatic cell” refers to any cell in the bodyother than gametes (egg or sperm); sometimes referred to as “adult”cells.

As used herein, the term “neural lineage cell” refers to a cell thatcontributes to the nervous system (both central and peripheral) orneural crest cell fates during development or in the adult. The nervoussystem includes the brain, spinal cord, and peripheral nervous system.Neural crest cell fates include cranial, trunk, vagal, sacral, andcardiac, giving rise to mesectoderm, cranial cartilage, cranial bone,thymus, teeth, melanocytes, iris pigment cells, cranial ganglia, dorsalroot ganglia, sympathetic/parasympathetic ganglia, endocrine cells,enteric nervous system, and portions of the heart.

As used herein, the term “adult stem cell” refers to a somatic stemcell, for one example, a “hematopoietic stem cell” which refers to astem cell in babies, children and adults, that gives rise to all red andwhite blood cells and platelets.

As used herein, the term “embryonic stem cell” refers to a primitive(undifferentiated) cell that is derived from one of several sources,including but not limited to a preimplantation-stage embryo, anartificially created embryo, i.e. by in vitro fertilization, etc.,capable of dividing without differentiating for a prolonged period inculture, and are known to have the capability to develop into cells andor tissues of the three primary germ layers, the ectoderm, the mesoderm,and the endodeiui.

As used herein, the term “endoderm” refers to a layer of the cellsderived from the inner cell mass of the blastocyst; it has thecapability to give rise to lungs, other respiratory structures, anddigestive organs, or generally “the gut” “in vivo” and a variety of celltypes in vitro.

As used herein, the term “embryonic stem cell line” refers to apopulation of embryonic stem cells that have been cultured under invitro conditions that allow proliferation without differentiation for upto days, months to years, for example, cells in a human WA-09 cell line.

As used herein, the term “human embryonic stem cell” or “hESC” refers toa type of pluripotent stem cells derived from early stage human embryos,up to and including the blastocyst stage, that is capable of dividingwithout differentiating for a prolonged period in culture, and are knownto develop into cells and tissues of the three primary germ layers, theectoderm, the mesoderm, and the endoderm.

As used herein, the term “induced pluripotent stem cell” or “iPSC”refers to a type of pluripotent stem cell, similar to an embryonic stemcell, whereby somatic (adult) cells are reprogrammed to enter anembryonic stem cell-like state by being forced to express factorsimportant for maintaining the “sternness” of embryonic stem cells(ESCs). Mouse iPSCs were reported in 2006 (Takahashi and Yamanaka), andhuman iPSCs were reported in late 2007 (Takahashi et al. and Yu et al.).Mouse iPSCs demonstrate important characteristics of pluripotent stemcells, including the expression of stem cell markers, the formation oftumors containing cells from all three germ layers, and the ability tocontribute to many different tissues when injected into mouse embryos ata very early stage in development. Human iPSCs also express stem cellmarkers and are capable of generating cells characteristic of all threegerm layers. Unlike an embryonic stem cell an iPSC is formedartificially by the introduction of certain embryonic genes (such as aOCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi andYamanaka Cell 126, 663-676 (2006), herein incorporated by reference)into a somatic cell, for examples of cell lines from induced cells, C14,C72, and the like. Another example of an iPSC is an adult human skincell, or fibroblast cell, transformed with using genes (OCT4, SOX2,NANOG, L1N28, and KLF4) cloned into a plasmid for example, see, Yu, etal., Science DOI: 10.1126/science.1172482, herein incorporated byreference.

As used herein, the term “totipotent” refers to an ability to give riseto all cell types of the body plus all of the cell types that make upthe extraembryonic tissues such as the placenta.

As used herein, the term “multipotent” refers to an ability to developinto more than one cell type of the body.

As used herein, the term “pluripotent” refers to a cell having theability to give rise to at least two but often numerous different celltypes of the body. Pluripotent cells often generate a teratoma afterinjection into an immunosuppressed mouse.

As used herein, the term “pluripotent stem cell” refers to an ability ofthis cell to develop into at least two different cells types dependingupon environmental factors, i.e. morphogcns, growth factors, signalingmolecules, either activators or inhibitors, etc. In some embodiments, apluripotent stem cell refers to an ability of a cell to develop into anyone of the three developmental germ layers of the organism includingendoderm, mesoderm, and ectoderm.

As used herein, the term “specialized cell” refers to a type of cellthat performs a specific function in multicellular organisms. Forexample, groups of specialized cells, such as neurons, work together toform a system, such as a nervous system.

As used herein, the term “neuroectoderm” refers to a cell or cell fatefound early in development or during pluripotent stem celldifferentiation that can give rise to cells of the neural lineage.

As used herein, the term “markers of cell proliferation” refers to theexpression of molecules associated with rapidly cycling cells which aretypically not present in mature slowly cycling or noncycling cells, i.e.actively dividing vs. cells with extended cycling times or noncyclingcells. Examples of such markers include a Ki67 marker of cellproliferation (Gerdes, et al., Int J Cancer 31:13-20 (1983), hereinincorporated by reference) and phospho-histone H3 markers of G2/M-phasesof mitosis (Hendzel, et al., Chromosoma 106:348-360 (1997), hereinincorporated by reference).

As used herein, the term “proliferation” refers to an increase in cellnumber.

As used herein, the term “differentiation” refers to a process wherebyan unspecialized embryonic cell acquires the features of a specializedcell such as a specific type of neuron, brain cell, heart, liver, ormuscle cell. Differentiation is controlled by the interaction of acell's genes with the physical and chemical conditions outside the cell,usually through signaling pathways involving proteins embedded in thecell surface.

As used herein, the term “differentiation” as used with respect to cellsin a differentiating cell system refers to the process by which cellsdifferentiate from one cell type (e.g., a multipotent, totipotent orpluripotent differentiable cell) to another cell type such as atarget-differentiated cell.

As used herein, the term “cell differentiation” refers to a pathway bywhich a less specialized cell (i.e. stem cell) develops or matures topossess a more distinct form and function (for example, an iPSCprogressing into a neural crest progenitor to a cell of neuronal lineageto a floor plate midbrain progenitor cells to a midbrain fateFOXA2/LMX1A+ dopamine (DA) neurons of the present inventions).

As used herein, the term “undifferentiated” refers to a cell that hasnot yet developed into a specialized cell type.

As used herein, the term “default” or “passive” in reference to a celldifferentiation pathway refers to a pathway where a less specializedcell becomes a certain differentiated cell type in culture, when nottreated with certain compounds i.e. normal cell cultures conditionswithout contact with at least one morphogen. In other words, a defaultcell results when a cell is not contacted by a molecule capable ofchanging the differentiated cell type (i.e. a morphogen), for examplecultures treated with LSB alone, but not an activator of SHH or anactivator of Wnt for making a forkhead box protein A2 (FOXA2)+ cell ofthe present inventions, instead results in the expression of markersHES5, PAX6, LHX2, and EMX2. In contrast, “non-default” in reference to acell refers to a differentiated cell type that results in a cell typethat is different from a default cell, i.e. a non-default cell is adifferentiated cell type resulting from a non-default conditions, suchas cell of the present inventions, including a forkhead box protein A2(FOXA2)+ neuronal cell, a floor plate midbrain progenitor cell andmidbrain fate FOXA2/LMX1A+ dopamine (DA) neuron of the presentinventions, etc. A default cell may also be a default cell after a cellhas contact with a morphogen to become a non-default cell without asubsequent morphogenic compound, such as a non-default floor platemidbrain progenitor cell that subsequently becomes a default cell thatis not a midbrain fate FOXA2/LMX1A+ dopamine (DA) neurons because of alack of contact with a morphogen such as CHIR.

As used herein, the term “morphogen” refers to a compound thatinfluences differentiation of a cell, i.e. determines, at least in part,cell fate. A morphogen also can influence a cell to differentiate into anon-default cell type.

As used herein, the term “directed differentiation” refers to amanipulation of stem cell culture conditions to induce differentiationinto a particular (for example, desired) cell type, such as floor platemidbrain progenitor cells and midbrain fate FOXA2/LMX1A+ dopamine (DA)neurons of the present inventions. In one embodiment, the term “directeddifferentiation” in reference to a cell refers to the use of smallmolecules, growth factor proteins, and other growth conditions topromote the transition of a cell from a pluripotent state into a moremature or specialized cell fate (e.g. central nervous system cell,neural cell, floor plate midbrain progenitor cell and midbrain fateFOXA2/LMX1A+ dopamine (DA) neuron of the present inventions, etc.). Inone preferred embodiment, the beginning of directed differentiation isthe contacting of a cell at day 0 with LDN/SB. A cell undergoingdirected differentiation as described herein results in the formation ofa non-default cell type of floor plate midbrain progenitor cells andmidbrain fate FOXA2/LMX1A+ dopamine (DA) neurons of the presentinventions.

As used herein, the term “inducing differentiation” in reference to acell refers to changing the default cell type (genotype and/orphenotype) to a non-default cell type (genotype and/or phenotype). Thus“inducing differentiation in a stem cell” refers to inducing the cell todivide into progeny cells with characteristics that are different fromthe stem cell, such as genotype (i.e. change in gene expression asdetermined by genetic analysis such as a microarray) and/or phenotype(i.e. change in expression of a protein, such as forkhead box protein A2(FOXA2) or a set of proteins, such as forkhead box protein A2 (FOXA2)and LIM homeobox transcription factor 1, alpha (LMX1A) positive (+)while negative (−) for PAX6).

As used herein, the term “fate” in reference to a cell, such as “cellfate determination” in general refers to a cell with a geneticallydetermined lineage whose progeny cells are capable of becoming a varietyof cell types or a few specific cell types depending upon in vivo or invitro culture conditions. In other words, a cell's predetermined fate isdetermined by its environment to be destined for a particulardifferentiation pathway such that a cell becomes one cell type insteadof another cell type, for example, a stem cell's progeny cells whose“neural fate” is to become a nerve cell instead of a muscle cell or askin cell.

As used herein, the term “neurite outgrowth” or “neural outgrowth”refers to observation of elongated, membrane-enclosed protrusions ofcytoplasm from cells.

As opposed to “neural overgrowth” which refers to unwanted unconstrainedneural growth, i.e. uncontrolled growth of neurons, of transplantedcells at the site of engraftment. As used herein, the term “teratoma”refers to a noncancerous tumour from any tissue type growing fromtransplanted cells.

As used herein, the term “teratoma formation” refers to the unwantedgrowth of a variety of tissue types into noncancerous tumours fromgrowth of transplanted cells.

As used herein, the term “dopamine neuron” or “dopaminergic neuron” ingeneral refers to a cell capable of expressing dopamine. “Midbraindopamine neurons” or “mDA” refer to presumptive dopamine expressingcells in forebrain structures and dopamine expressing cells in forebrainstructures.

As used herein, the term “neural stem cell” refers to a stem cell foundin adult neural tissue that can give rise to neurons and glial(supporting) cells. Examples of glial cells include astrocytes andoligodendrocytes.

As used herein, the term “floor plate” or “FP” or “fp” refers to aregion of the neural tube in vivo that extends along the entire ventralmidline also described as the unpaired ventral longitudinal zone of theneural tube or referred to as a signaling center of the neural tube. Inother words, the neural tube was divided in different regions where theventral cells closest to the midline constituted the floor plate. Forone example of further cellular identification, chick midbrain FP can bedivided into medial (MFP) and lateral (LFP) regions on the basis of geneexpression, mode of induction and function. Floor plate cells are foundin vivo in several areas of the developing embryo, for example floorplate cells in the midbrain, in the hindbrain, etc. In vivo, floor platecells in the midbrain region are contemplated to give rise to cells thatare different than cells differentiated from floor plate cells in otherregions. One primary floor plate marker in the midbrain region is FOXA2.

As used herein, the term “roof plate” refers to the dorsal cells closestto the midline. One roof plate marker is LMX1A. During embryonicdevelopment, floor plate and roof plate cells are located at distinctpositions in the CNS (ventral versus dorsal) with diametrically opposedpatterning requirements for their induction.

As used herein, the term “midbrain” refers to a region of the developingvertebrate brain between the forebrain (anterior) and the hindbrain(posterior). The midbrain regions gives rise to many areas of the brain,including but not limited to reticular formation, which is part of thetegmentum, a region of the brainstem that influences motor functions,the crus cerebri, which is made up of nerve fibers connecting thecerebral hemispheres to the cerebellum, and a large pigmented nucleuscalled the substantia nigra. A unique feature of the developing midbrainis the co-expression of the floor plate marker FOXA2 and the roof platemarker LMX1A.

As used herein, the term “neuron” refers to a nerve cell, the principalfunctional units of the nervous system. A neuron consists of a cell bodyand its processes-an axon and one or more dendrites. Neurons transmitinformation to other neurons or cells by releasing neurotransmitters atsynapses.

As used herein, the term “cell culture” refers to a growth of cells invitro in an artificial medium for research or medical treatment.

As used herein, the term “culture medium” refers to a liquid that coverscells in a culture vessel, such as a Petri plate, a multiwell plate, andthe like, and contains nutrients to nourish and support the cells.Culture medium may also include growth factors added to induce desiredchanges in the cells.

As used herein, the term “neuronal maturation medium” or “BAGCT” mediumrefers to a culture medium comprising N2 medium, further comprisingbrain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cellline-derived neurotrophic factor, dibutyryl cAMP and transforming growthfactor type ß3 for differentiating midbrain fate FOXA2/LMX1A+ dopamine(DA) neurons.

As used herein, the term “feeder layer” refers to a cell used inco-culture to maintain pluripotent stem cells. For human embryonic stemcell culture, typical feeder layers include mouse embryonic fibroblasts(MEFs) or human embryonic fibroblasts that have been treated to preventthem from dividing in culture.

As used herein, the term “passage” in reference to a cell culture,refers to the process in which cells are disassociated, washed, andseeded into new culture vessels after a round of cell growth andproliferation. The number of passages a line of cultured cells has gonethrough is an indication of its age and expected stability.

As used herein, the term “expressing” in relation to a gene or proteinrefers to making an mRNA or protein which can be observed using assayssuch as microarray assays, antibody staining assays, and the like.

As used herein, the term “paired box gene 6” or “PAX6” refers to amarker of a non-default neuroprogenitor cell.

As used herein, the term “TUJ1” or “neuron-specific class IIIbeta-tubulin” in reference to a differentiating cell of the presentinventions refers to a marker of early neural human celldifferentiation, such as neural progenitor cells, and is found expressedin neurons of the PNS and CNS.

As used herein, the term “homodimer” in reference to a SMAD moleculerefers to at least two molecules of SMAD linked together, such as bydisulfide linkages.

As used herein, the term “contacting” cells with a compound of thepresent inventions refers to placing the compound in a location thatwill allow it to touch the cell in order to produce (obtain) “contacted”cells. The contacting may be accomplished using any suitable method. Forexample, in one embodiment, contacting is by adding the compound to atube of cells. Contacting may also be accomplished by adding thecompound to a culture of the cells.

As used herein, the term “attached cell” refers to a cell growing invitro wherein the cell adheres to the bottom or side of the culturevessel, an attached cell may contact the vessel via extracellular matrixmolecules and the like and requires the use of an enzyme for detachingthis cell from the culture dish/container, i.e. trypsin, dispase, etc.An “attached cell” is opposed to a cell in a suspension culture that isnot attached and does not require the use of an enzyme for removingcells from the culture vessel.

As used herein, the term “marker” or “cell marker” refers to a gene orprotein that identifies a particular cell or cell type. A marker for acell may not be limited to one marker, markers may refer to a “pattern”of markers such that a designated group of markers may identity a cellor cell type from another cell or cell type. For example, midbrain fateFOXA2/LMX1A+ dopamine (DA) neurons of the present inventions express oneor more markers that distinguish a floor plate midbrain progenitor cellfrom a precursor less differentiated cell, i.e. forkhead box protein A2(FOXA2) positive and LIM homeobox transcription factor 1, alpha (LMX1A)positive vs. HES5+ and PAX6+ cells, for example, as shown by exemplarygene expression patterns in FIGS. 1e and 1 f.

As used herein, the term “positive” in relation to a cell, including a“positive cell” refers to a cell that expresses a marker, for oneexample, an antibody “stains” for that marker when using an antibodystaining (detection) system or a nucleic acid sequence that hybridizesto the marker nucleic acid sequence as measured by a reporter molecule,i.e. a fluorescent molecule that attaches to double stranded nucleicacid sequences, in a detectable quantitative and/or qualitative amountabove a control or comparative cell. For example, a cell positive for amarker such as forkhead box protein A2 (FOXA2), etc., refers to a cellthat expresses FOXA2 mRNA and/or protein when detected in an assay, suchas a gene array or antibody, respectively. Such as positive cell may bereferred to as FOXA2+. When a cell is positive for more than one marker,such as when using the notation FOXA2/LMX1A+, the cell or the majorityof the cell population is positive for both FOXA2 and LMX1A.

As used herein, the term “negative” in relation to a cell or cellpopulation, including a “negative cell” refers to a cell or populationabsent detectable signal for a marker or signal at levels of controlpopulations. For example, a cell failing to stain following contactingwith a forkhead box protein A2 (FOXA2) antibody detection method or genearray that includes detection of a FOXA2 mRNA, etc., is FOXA2- ornegative for FOXA2.

As used herein, the terms “reporter gene” or “reporter construct” referto genetic constructs comprising a nucleic acid encoding a protein thatis easily detectable or easily assayable, such as a colored protein,fluorescent protein such as GFP or an enzyme such as β-galactosidase(lacZ gene).

As used herein, the term “GFP” refers to any green fluorescent proteinDNA sequence capable of producing a fluorescent protein upon expressionin a cell typically used as an indication marker for expression of atarget gene. Examples of GFP include GFP sequences isolated fromcoelenterates, such as the Pacific jellyfish, Aequoria Victoria, andsynthetic sequence derivatives thereof, such as “eGFP”.

The term “sample” is used in its broadest sense. In one sense it canrefer to a cell or tissue. In another sense, it is meant to include aspecimen or culture obtained from any source and encompasses fluids,solids and tissues. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

The terms “purified,” “to purify,” “purification,” “isolated,” “toisolate,” “isolation,” and grammatical equivalents thereof as usedherein, refer to the reduction in the amount of at least one contaminantfrom a sample. For example, a desired cell type is purified by at leasta 10%, preferably by at least 30%, more preferably by at least 50%, yetmore preferably by at least 75%, and most preferably by at least 90%,with a corresponding reduction in the amount of undesirable cell types,for example, directed differentiation of the present inventions resultedin the desired increase in purity of differentiated floor plate midbrainprogenitor cells or midbrain fate FOXA2/LMX1A+ dopamine (DA) neurons ofthe present inventions. In other words “purify” and its equivalents,refers to the removal of certain cells (e.g., undesirable cells) from asample either mechanically, such as by flow cytometer cell sorting orthrough directed differentiation. For example, for differentiating apurified population of forkhead box protein A2 (FOXA2)+ LIM homeoboxtranscription factor 1, alpha (LMX1A)+ progenitor cells of the presentinventions, progenitor cells are purified by removal of contaminatingPAX6 neuronal cells by sorting a mixed cell population into doublepositive forkhead box protein A2 (FOXA2)+ LIM homeobox transcriptionfactor 1, alpha (LMX1A)+ cells by flow cytometry; midbrain fateFOXA2/LMX1A+ dopamine (DA) neurons are also purified or “selected” fromnon-dopamine (DA) (default cells) by using a specified method of cellculture comprising compositions and methods of the present inventions.The removal or selection of non-midbrain fate FOXA2/LMX1A+ dopamine (DA)neuronal cells results in an increase in the percent of desired midbrainfate FOXA2/LMX1A+ dopamine (DA) neurons in the sample. Thus,purification of a cell type results in an “enrichment,” i.e., anincrease in the amount, of the desired cell, i.e. midbrain fateFOXA2/LMX1A+ dopamine (DA) neurons in the sample.

The term “naturally occurring” as used herein when applied to an object(such as cell, tissue, etc.) and/or chemical (such as a protein, aminoacid sequence, nucleic acid sequence, codon, etc.) means that the objectand/or compound are/were found in nature. For example, a naturallyoccurring cell refers to a cell that is present in an organism that canbe isolated from a source in nature, such as an embryonic cell, whereinthe cell has not been intentionally modified by man in the laboratory.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments exemplified, but are not limited to,test tubes and cell cultures.

As used herein the term, “in vivo” refers to the natural environment(e.g., an animal or a cell) and to processes or reactions that occurwithin a natural environment, such as embryonic development, celldifferentiation, neural tube formation, etc.

The term “derived from” or “established from” or “differentiated from”when made in reference to any cell disclosed herein refers to a cellthat was obtained from (e.g., isolated, purified, etc.) a parent cell ina cell line, tissue (such as a dissociated embryo, or fluids using anymanipulation, such as, without limitation, single cell isolation,cultured in vivo, treatment and/or mutagenesis. A cell may derived fromanother cell, using for example chemical treatment, radiation, inducingnew protein expression, for example, by infection with virus,transfection with DNA sequences, contacting (treating) with a morphogen,etc., and selection (such as by serial culture) of any cell type that iscontained in cultured parent cells). A derived cell can be selected froma mixed population by virtue of response to a growth factor, cytokine,selected progression of cytokine treatments, adhesiveness, lack ofadhesiveness, sorting procedure, and the like.

As used herein, the term “cell” refers to a single cell as well as to apopulation of (i.e., more than one) cells. The population may be a purepopulation comprising one cell type, such as a population of neuronalcells or a population of undifferentiated embryonic cells.Alternatively, the population may comprise more than one cell type, forexample a mixed cell population. It is not meant to limit the number ofcells in a population; for example, in one embodiment, a mixedpopulation of cells may comprise at least one differentiated cell. Inthe present inventions, there is no limit on the number of cell typesthat a cell population may comprise.

As used herein, the term “highly enriched population” refers to apopulation of cells, such as a population of cells in a culture dish,expressing a marker at a higher percentage or amount than a comparisonpopulation, for example, treating a LSB contacted cell culture on day 1with purmorphamine and on day 3 with CHIR results in a highly enrichedpopulation of floor plate midbrain progenitor cells compare to treatmentwith LSB alone. In other examples, an enriched population is apopulation resulting from sorting or separating cells expressing one ormore markers from cells not expressing the desired marker, such as aCD142 enriched population, an A9 enriched population, and the like.

The term, “cell biology” or “cellular biology” refers to the study of alive cell, such as anatomy and function of a cell, for example, a cell'sphysiological properties, structure, organelles, and interactions withtheir environment, their life cycle, division and death.

The term “nucleotide sequence of interest” refers to any nucleotidesequence (e.g., RNA or DNA), the manipulation of which may be deemeddesirable for any reason (e.g., treat disease, confer improvedqualities, expression of a protein of interest in a host cell,expression of a ribozyme, etc.), by one of ordinary skill in the art.Such nucleotide sequences include, but are not limited to, codingsequences of structural genes (e.g., reporter genes, selection markergenes, oncogenes, drug resistance genes, growth factors, etc.), andnon-coding regulatory sequences which do not encode an mRNA or proteinproduct (e.g., promoter sequence, polyadenylation sequence, terminationsequence, enhancer sequence, etc.).

As used herein, the term “protein of interest” refers to a proteinencoded by a nucleic acid of interest.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of apolypeptide or precursor (e.g., proinsulin). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The term also encompasses thecoding region of a structural gene and includes sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ untranslated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ untranslated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene that are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” “DNA encoding,” “RNA sequence encoding,” and “RNAencoding” refer to the order or sequence of deoxyribonucleotides orribonucleotides along a strand of deoxyribonucleic acid or ribonucleicacid. The order of these deoxyribonucleotides or ribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA or RNA sequence thus codes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of cell differentiation, a kit mayrefer to a combination of materials for contacting stem cells, suchdelivery systems include systems that allow for the storage, transport,or delivery of reaction reagents from one location to another in theappropriate containers (such as tubes, etc.) and/or supporting materials(e.g., buffers, written instructions for performing celldifferentiation, etc.) (e.g., compounds, proteins, detection agents(such as antibodies that bind to tyrosine hydroxylase (TH), forkhead boxprotein A2 (FOXA2), LIM homeobox transcription factor 1, alpha (LMX1A),etc.), etc. For example, kits include one or more enclosures (e.g.,boxes, or bags, test tubes, Eppendorf tubes, capillary tubes, multiwellplates, and the like) containing relevant reaction reagents forinhibiting signaling pathways, for example, an inhibitor for loweringtransforming growth factor beta (TGFβ)/Activin-Nodal signaling, such asSB431542 (or SB431542 replacement), and the like, an inhibitor forlowering SMAD signaling, LDN-193189 (or LDN-193189 replacement), and thelike, an inhibitor for lowering glycogen synthase kinase 3β (GSK3β), forone example, for activation of wingless (Wnt or Wnts) signalingotherwise known as a WNT signaling activator (WNT agonist), such asCHIR99021 (or CHIR99021 replacement), etc.), and the like, an activatorof Sonic hedgehog (SHH) signaling (such as a smoothened (SMO) receptorsmall molecule agonist), for example, a Sonic hedgehog (SHH) C25IImolecule, purmorphamine, and the like, a molecule with Fibroblast growthfactor 8 (FGF8) activity, such as Fibroblast growth factor 8 (FGF8),etc., and neuronal maturation molecules, for example, brain-derivedneurotrophic factor (BDNF), ascorbic acid (AA), glial cell line-derivedneurotrophic factor, dibutyryl cAMP and transforming growth factor typeß3, including molecules capable of replacing these components, and/orsupporting materials. The reagents in the kit in one embodiment may bein solution, may be frozen, or may be lyophilized. The reagents in thekit in one embodiment may be in individual containers or provided asspecific combinations, such as a combination of LSB (LDN-193189 withSB431542), Sonic hedgehog (SHH) C25II molecule with purmorphamine, Sonichedgehog (SHH) C25II molecule with purmorphamine with CHIR99021 orpurmorphamine with CHIR99021, neuronal maturation molecules and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary induction and neurogenic conversion of human EScell-derived midbrain floor plate precursors dependent on CHIR990221addition. a) Immunocytochemical analysis at day 11 of differentiationfor FOXA2 (red), NESTIN (green, upper panels), LMX1A (green, middlepanels) and OTX2 (green, lower panels) expression. b,c) Quantificationof the data presented in (a). Data are from three independentexperiments carried out each in triplicates (mean±SEM). Significancelevels for individual markers are presented as compared to LSB onlytreatment: ANOVA; Dunnett test: *** p<0.001; ** p<0.01; p<0.05). d)Schematic illustration of the culture conditions used for the threetreatment conditions. e,f) Lists of selected differentially expressedgenes at day 11 comparing LSB/S/F8/CHIR conditions with either LSB (e)or LSB/S/F8 (f). g,h) Temporal gene expression analysis of selectedmarkers characteristic of midbrain DA precursor identity (g), forebrainand ventral non-DA precursor identity (h). Scale bars correspond to 50μm.

FIG. 2 shows an exemplary immunocytochemical and molecular analysis ofmidbrain DA neuron fate in LSB/S/F8/CHIR treated versus LSB/S/F8(ventral/hypothalamic) and LSB (dorsal forebrain) fates. a)Immunocytochemical analysis at day 25 for expression of FOXA2(blue) incombination with Tuj1(red)/LMX1A(green) (upper panels) andNURR1(red)/TH(green) (lower panels). b) Quantitative co-expressionanalysis in LSB/S/F8/CHIR treated cultures. Data are from threeindependent experiments carried out each in triplicates (mean±SEM). c,d)Global gene expression analysis was performed at day 25 (triplicatesamples for all three conditions). Selected lists of the mostdifferentially expressed genes comparing day 13 versus day 25 in theLSB/S/F8/CHIR condition (c) and comparing LSB/S/F8/CHIR treatment versusLSB (d, left panel) and LSB/S/F8 (d, right panel). e) Normalizeddifferential gene expression analysis for key midbrain DA neuronmarkers. Significance levels for individual markers are presented ascompared to LSB only treatment: ANOVA; Dunnett test: *** p<0.001; **p<0.01; p<0.05). Scale bars correspond to 50 μm.

FIGS. 3-1 and 3-2 shows an exemplary in vitro maturation,characterization and functional assessment of floor plate derived-versusrosette-derived midbrain DA neurons. FIG. 3-1: shows exemplary: a)Immunocytochemical analysis at day 50 of differentiation for TH (red),in combination with LMX1A (green, left panels), FOXA2 (blue, leftpanels) and NURR1 (green, right panels). b) Quantification of TH+,FOXA2+, LMX1+ and NURR1+ cells out of total cells in rosette-derivedversus floor plate-derived (LSB/S/F8/CHIR) cultures. c) Quantificationof serotonin+(5-HT), and GABA+ neuronal subtypes at day 50 in floorplate- and rosette-derived DA neuron cultures. d,e) HPLC analysis formeasuring DA and metabolites d) Representative chromatogram for theelectrochemical detection of DA in a sample of floor plate-derivedcultures. e) Comparison of DA, DOPAC and HVA levels between floorplate-versus rosette-derived cultures. f) Immunocytochemical analysis offloor plate-derived cultures (day 80) for TH (red) and synapsin (green).g-i) Electrophysiological analyses of floor plate cultures at day 80 ofDA neuron differentiation. Phase contrast image of a patched neuron (g)and corresponding recordings (h). i) Power analysis showing membranepotential oscillations characteristic of DA neuron identity (2 toapproximately 5 Hz). Significance levels for individual markers (panelsb, c, e) are presented as a comparison of FP—versus rosette-derivedcultures: Student's T-test: *** p<0.001; ** p<0.01; p<0.05). Scale barscorrespond to 50 μm in (a), 20 μm in (f, upper panel), 5 μm in (f; lowerpanel) and 20 μM in (g) j: Maturation of mDA neurons in vitro (d65). THpositive neurons are still expressing FoxA2 and extend long fiberstypical for mDA neurons, and k: DA release measurement by HPLC: d65 oldTH+ neurons are functional in vitro. FIG. 3-2: shows an exemplarysummary of cells produced by a floor plate based midbrain DA neuronprotocol as described herein. a) In contrast to past strategies (forexample, Perrier, A. L. et al. Derivation of midbrain dopamine neuronsfrom human embryonic stem cells. Proc Natl Acad Sci USA 101, 12543-8(2004)), the novel protocol described herein is based on generatingLMX1A/FOXA2 positive midbrain floor plate (left panel) followed byneuronal conversion (middle panel) and DA neuron maturation (rightpanel). Mature floor plate generated DA neuron cells retain FOXA2/LMX1Aexpression.

FIG. 4 shows an exemplary in vivo survival and function of floorplate-derived human DA neurons in mouse, rat and monkey PD model hostbrain. a-d) Transplantation of floor plate-derived DA neurons in 6-OHDAlesioned adult mice (NOD-SCID IL2Rgc null strain). a) TH expression andgraft morphology at 4.5 months after transplantation. b) Expression ofhuman specific marker (hNCAM, blue), TH (green), and FOXA2 (red). c)Quantification of FOXA2+, TH+ and double-labeled cells in floorplate-derived grafts (mean±SEM, n=4 at 4.5 months post grafting). d)Amphetamine-induced rotation analysis in floor plate-derived (blue)versus rosette-derived (green) grafts. Scale bars correspond to 500 μmin (a), 100 μm in (b) and 40 μm in (4c). e-p) Transplantation of floorplate-derived DA neurons into 6-OHDA lesioned adult rats.Immunohistochemical analysis for co-expression of TH (green) and thehuman specific markers (red) hNA. e) and hNCAM (4f). g) Stereologicalquantification of the number of total (hNA+) cells, TH+ cells and TH+cells co-expressing FOXA2. The average graft volume was 2.6+/−0.6 mm³).h-j) High power images showing co-expression of TH (green) with midbrainspecific transcription factors FOXA2, PITX3 and NURR1 (red). k-m)Behavioral analysis of animals treated with floor plate-derived DAneuron grafts versus sham-treated animals. k) Amphetamine-inducedrotational asymmetry. l) stepping test: measuring forelimb akinesia inaffected versus non-affected side. m) Cylinder test: measuringipsilateral versus contra-lateral paw preference upon rearing. Graftedanimals showed significant improvement in at least three tests (p<0.01at 4.5-5 month; n=4-6 each). n-p) Immunohistochemical analysis for TH(green) and co-expression (red) with DAT (n), GIRK2 (o) and calbindin(p). Significance levels (panels d, k, 1, m) are: ** p<0.01; p<0.05).Scale bars correspond to 200 μm in (e), 50 μm in (f), 20 μm in (h j) and40 μm in (n-p). q-t) Transplantation of floor plate-derived DA neuronsinto adult 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesionedrhesus monkey. q) Overview of representative graft site at 1 month aftertransplantation marked by expression of human specific cytoplasm markerSC-121 (green). r) TH expression in graft with a surrounding halo of TH+fibers (arrows). s) Analysis of co-expression of SC-121 (red) with TH(green) in graft core. t) Co-expression analysis of FOXA2 (red) in TH+neurons (green). Scale bars correspond to 2 mm for (q), 500 μm for (r),200 μm for (s), and 50 μm for (t). u) graft derived fiber outgrowth(huNCAM+) into the host striatum. v) Graft-derived cells do notdifferentiate into glial cell. However, grafts contained a fewserotonergic fibers in addition to the TH+ cell population.

FIG. 5 shows an exemplary timing of CHIR99021 exposure determinesinduction of FOXA2/LMX1A midbrain floor plate precursors. a)Immunocytochemical analysis of FOXA2(red)/LMX1A(green) at day 11 ofdifferentiation following LSB/S/F8 treatment alone or in combinationwith CHIR starting at the various time points indicated. b)Quantification of the percentage of FOXA2+, LMX1A+ and double labeledcells at day 11 of differentiation following differential onset of CHIRexposure as described in (a). Significance levels for individual markersare presented as compared to no CHIR condition: ANOVA; Dunnett test: ***p<0.001; ** p<0.01; p<0.05). Scale bars correspond to 50 μm.

FIG. 6 shows an exemplary FGF8 exposure does not play a major role inthe induction of FOXA2/LMX1A midbrain floor plate precursors.Representative images of FOXA2(red)/LMX1A(green) expression byimmunocytochemistry at day 11 of differentiation. Cells were exposed toLSB/CHIR in the presence or absence of SHH (purmorphamine+SHH C25II) andFGF8. Scale bars correspond to 50 μm.

FIG. 7 shows an exemplary exposure to high dose of SHH and/or asmoothened small molecule agonist (purmorphamine) is required forefficient midbrain floor plate induction in the presence of CHIR99021.Representative images of FOXA2 (red)/LMX1A (green) immunocytochemistryat day 11 of differentiation. Cells were treated with LSB/F8/CHIR in thepresence of various concentrations of SHH (SHH-C25II) and smoothenedagonist purmorphamine Scale bars correspond to 50 μm.

FIG. 8 shows an exemplary analysis of genes differentially expressed inLSB/S/F8/CHIR treated versus LSB and LSB/S/F8 treated cultures at days11 and 25 of differentiation. a) Hierarchical clustering of the globalgene expression data obtained from three conditions at days 0, 1, 3, 5,7, 11 and 13 (samples were assessed in triplicates for each conditionand each day). b) Gene enrichment analysis according to GO classes usingDAVID; http://david.abcc.ncifcrf.gov. Comparisons at day 11 revealenrichment for both SHH and WNT signaling in LSB/S/F8/CHIR condition inagreement with CHIR99021 mediated activation of canonical WNT signaling.Alternative cell fates such as “forebrain development” and“diencephalon” as well as “homeobox” are other GO term's highly enrichedat days 11 and 25. c) Online validation using the Allen gene humanexpression data base (http://human.brain-map.org) for candidate markersenriched in midbrain DA neuron condition (LSB/S/F8/CHIR). TTF3, EBF1,EBF3 and TTR were expressed based on available human brain regionspecific microarray data. TTR, a classic transcriptional target of FOXA2is only weakly expressed in adult substantia nigra, suggesting that itsmain role may be during development or that SHH treatment may causeartificially high TTR expression levels during in vitro differentiation.

FIG. 9 shows an exemplary differentiation protocol for floor plateinduction and midbrain DA neuron development in representativeindependent hESC and hiPSC lines. Data from the hESC line H1 and thehiPSC lines 2C6 (from a sporadic PD patient) and SeV6 (Sendai-based,integration-free) are presented. The floor plate based protocoldescribed in FIG. 1d and FIG. 10, was used followed by analysis of FOXA2(red) expression at day 11 and TH(green)/FOXA2(red) at day 25 ofdifferentiation.

FIG. 10 shows an exemplary schematic summary of the differentiationconditions used for floor plate-derived and rosette-derived DA neuroncultures. Both protocols used dual-SMAD inhibition to accelerate neuralfate acquisition. LDN was used for BMP inhibition in the floor plateprotocol while the traditional noggin induction was used for rosettecultures. The abbreviations are: LDN: LDN-193189, SB: SB431542, SHH(purmorphamine+SHH C25II), FGF8: FGF8, BAGCT: BDNF+ascorbicacid+GDNF+dbcAMP+TGFβ3. SHH/FGF8 in the rosette protocol used SHH C25Ialone in the absence of purmorphamine following the initialrecommendation for patterning of rosette-derived DA neuron cultures.Note: Purmorphamine treatment at rosette stage shows toxicity atconcentration suitable for patterning floor plate cells. BASF:BDNF+ascorbic acid+SHH/FGF8.

FIG. 11 shows an exemplary in vitro maturation of floor plate-derived DAneuron cultures. a) Immunocytochemical analysis of floor plate-derivedTH neurons (green) for DAT expression (red; day 80). b) ExtensiveTuj1+(red) fiber tracts were observed by day 60 of differentiationextending over distances of >2 mm. c) At day 80 of differentiation,co-expression of GIRK2 (red) in TH+ neurons (green). Scale barscorrespond to 20 μm in (a) 100 μm in (b) and 20 μm in (c).

FIG. 12 shows an exemplary immunohistochemical analysis of short-term (6weeks) in vivo survival studies in adult intact (unlesioned) mousestriatum (NOD-SCID IL2Rgc null strain). Analysis of floor plate derivedgrafts (day 25 cells; 150×10³ cells/animal). a) Representative image ofgraft core showing TH+ cells surrounded by TH+host fibers. An average of6,200 TH+ cells (n=3) were present in the graft at 6 weeks aftertransplantation. b) FOXA2 expressing cells (red) were only found in thegraft but not in the surrounding host striatum demonstrating graftorigin and midbrain identity of the cells. Nearly all TH+ neurons(green) co-expressed FOXA2 (red). However, a considerable proportion ofFOXA2+ cells did not co-express TH suggesting that these cells have notyet acquired a mature DA phenotype or represent another FOXA2+ neuronalpopulation negative for DA neuron markers. Scale bars correspond to 50μm.

FIG. 13 shows an exemplary histological analysis of long-term (4.5months) grafted 6-OHDA lesioned mice (NOD-SCID IL2Rgc null strain)comparing behavior of floor plate-versus rosette-derived grafts.Analysis of floor plate-derived grafts. a) Example of one of the largestfloor plate-derived grafts. The graft retains a well circumscribedhNCAM+(red) cytoarchitecture. b) Robust hNCAM+ fiber outgrowth wasobserved at graft periphery. c) Serotonergic (5-HT+) fibers (green) ingraft are largely negative for hNCAM (red) suggesting host origin. d)GABAergic neurons and fibers (green) in graft. Analysis ofrosette-derived grafts: e) Neural overgrowth with compression of hostbrain tissue. The majority of cells were positive for both NCAM (red)and DCX (green) suggesting neuronal fate. f) NCAM+/DCX+ fibers extendedto the non-transplanted contra-lateral side of the brain. g) Within thegraft core multiple DCX+ clusters were observed. h) Few TH+ cells(green) and fibers were observed at graft periphery and nearly allrosette-derived TH+ cells in vivo were negative for FOXA2 (blue).Irnmunohistochemical analysis of floor plate-derived versusrosette-derived grafts at 4.5 months. Representative images arepresented for expression of the proliferation marker Ki67 (red) and theneural precursor marker PAX6 (green) (i), expression of FOXA2/DCX (j),FOXG1/hNCAM (j, inset), and the astrocytes marker GFAP (green) (k).Scale bars correspond to 500 μm in (a), 50 μm in (b-d), to 500 μm in(e), 50 μm in (g-h), 50 μm in (i), 40 μm in (j) and 20 μm in (k).

FIG. 14 shows an exemplary histological analysis of long-term (5 months)grafted 6-OHDA lesioned SD rats. a) The large majority of hNA+ (red)cells expressed Tuj1 (green) indicating neuronal fate identity. b) GFAP+fibers (green) within the graft did not co-express hNA (red) suggestinghost origin. c) A few human serotonergic (5-HT+) cell bodies (green)were observed while most serotonergic fibers were host-derived similarto the data in mouse host (FIG. 13C). d) Additional example of TH+neurons (green) within graft characterized for expression of hNCAM (red)to confirm human identity of cells in host brain. Scale bars correspondto 25 μm in panels.

FIG. 15 shows an exemplary histological analysis of floor plate-derivedgrafts in primate brain. Floor plate-derived DA neurons derived from H9hESCs and from H9-GFP hESCs (day 25 cultures; 1.25×10⁶ cells/tract, 3tracts/per hemisphere, 6 tracts in total) were grafted into the striatumof adult MPTP lesioned rhesus monkeys (=2). a) Upper left and rightpanels: High-resolution image reconstructions of representative graftsites (1 month after transplantation). Graft cytoarchitecture isillustrated by immunohistochemistry for the human specific marker SC-121(no cross-reactivity with non-human primate tissues). Lower left panel:SC-121+ fibers extending from graft core. Lower right panel: Higherresolution image of SC-121+ cells showing neuronal precursormorphologies. b) Analysis of GFP expression in graft cores furtherconfirmed human identity of the cells complementing the SC-121 data.Note: For both animals, one hemisphere each was grafted with unmarked H9derived cells while the other hemisphere received cells derived fromH9-GFP cells (expression of GFP under control of EF1a promoter). c)Higher resolution image of the GFP+ graft using DAB detection showingnumerous GFP+ cells at graft periphery exhibiting neuroblast morphology.d) Immunohistochemical analyses of areas in the graft core negative forGFP and SC-121. These areas contained large numbers of host microgliabased on Iba1 expression (red) as well as few ED1+ macrophages (blue),suggesting persistent inflammation despite cyclosporineimmunosuppression. Scale bars correspond to 500 μm in (a, top panel), 50μm in (a, lower left panel), 20 μm in (a, lower right panel), 2 mm in(b), 500 μm in (c, left panel), 50 μm (c, upper right panel), 100 μm (c,lower right panel), 100 μm (d).

FIG. 16 shows an exemplary derivation of TH+ cells from hESCs using aprevious MS5 feeder cell based method that differentiated cells into DAneuron-like cells via (through) rosette cell intermediates. Top) at P0hESCs were contacted with molecules for beginning neural induction ofOct4+ cells into rosette cells using MS5 feeder cells (Perrier et al.,2004). At the P1 stage rosette cells were expanded by contacting cellswith additional molecules for differentiating cells into cells at stageP2 with specific expression patterns including Pax2+/En1+ DA progenitorcells further differentiated into TH+/En1+ DA neurons. These cells wereused for engraftment in 6OHDA lesion rats, immunosuppressed viacyclosporin A treatment. These transplantation studies showed very poorin vivo viability of DA-like neurons, including loss of the TH+phenotype, and revealed concerns about further growth of unwanted,possibly lethal, cells for the grafted animals, i.e. teratomas, andgrowth of cells into inappropriate neural types that would causeadditional medical problems for the patient. A: There were very smallnumbers of surviving TH+ neuron at 4.5 months after transplantation (<50TH+ cells/animal) in grafts from rosette derived DA neuron precursors.However, in contrast to TH+ cells, GFP marked cells (GFP was driven by aubiquitous promoter) did survive quite well after transplantation. Thissuggests that most surviving cells following transplantation were neuralcells of non-DA neuron identity (16B). FIG. 16C:few graft-derived cells(hNA+ (green) co-express TH (red) suggesting that most grafted humancells adopt a non-DA neuron phenotype. Panels 16 D-E show that D-E,despite the very poor in vivo survival there was some (albeit verymodest and highly variable) improvement in a few behavioral assays suchas amphetamine induced rotations (D), cylinder test and spontaneousrotations (E).

FIG. 17 shows an exemplary protocol for derivation of low numbers offloor plate cells. A modified Dual-SMAD inhibition protocol generatedfloor plate (fp) cells. High concentrations of SHH were necessary forthe induction of FoxA2+ fp cells and that addition of caudalizingpatterning cues such as FGF8, Wnt1 or RA did not lead to decrease inFOXA2 expression but change in regional identity A) Left panel: Cells atday 11 of differentiation following treatment with NSB(Noggin/SB431542); left center panel: NSB+SHH (Sonic C25I1) treatment;Center panel: NSB+SHH+RA; Center right pane: NSB+SHH+Wnt1; NSB+SHH+FGF8;Note: NSB only treatment does not induce FoxA2 expression. FoxA2+ floorplate cells are only induced in the presence of high dose SHH. Additionof RA. Wnt1 and FGF8 does not inhibit FoxA2 induction. B) qRT-PCRanalysis for gene expression of FOXA2 and SIX6 showing maintenance (oreven increase) of expression for FOXA2 following treatment with RA orFGF with a concomitant downregulation of SIX6 expression marking themost anterior floor plate like cells. C) Induction of gene expressionfor midbrain precursors and midbrain floor plate markers in the presenceof FGF8 and Wnt1.

FIG. 18 shows an exemplary protocol for derivation of floor plate cellsshowing high levels of midbrain characteristics as compared to the lowor absent levels in cells made from the procedures used in FIGS. 16 and17. A: Midbrain floor plate induction in cell populations contacted withhigh levels of SHH, FGF8 and CHIR resulted in FoxA2 co-expression withmidbrain markers LMX1A and Otx2 expression at day 11 of differentiationin contrast to cells contacted with molecules described in two otherprocedures as shown in FIGS. 16 and 17 (N/SB and SHH/FGF8,respectively). B: Global gene expression analysis at day 11 comparingthe three groups of cells contacted with molecules from each of thethree procedures including the third procedure of the present inventions(LDN/SB, SHH/FGF8, LSB/SHH/FGF8/CHIR) Chart B shows specifically thegenes that are common among the three treatment conditions versus thosegenes that are unique to each individual condition. Chart B is apreliminary analysis used for the microarray results presented in FIG.1.C: mRNA levels of midbrain markers FoxA2, LMX1A, are highly enrichedin LSB/SHH/FGF8/CHIR-treated group compared to SHH/FGF-treated group.

FIG. 19 shows an exemplary in vitro characterization of dopamine neuronsderived from the midbrain region of the floor plate. A: Co-labeling ofFoxA2+ neurons with mDA neuron markers TH, Nurr1 and LMX1A at d25 ofdifferentiation. B: mRNA expression levels by QRT-PCR of mDA neuronmarkers as well as other midbrain cell types inLDN/SB+SHH/FGF8+SHH/FGF8+CHIR treated groups.

FIG. 20 shows an exemplary comparable differentiation potential towardsmidbrain DA neuron fate of PINK1 mutant PD-iPS cells versus wild-typehES (or iPS) cells. PINK1 Q456X mutant PD-iPSC line was differentiatedusing the novel floor-plate based midbrain DA neurons methods of thepresent inventions yielding midbrain differentiation profiles comparableto those obtained from H9 line. A-C) Immunocytochemical analysis ofPINK1 mutant PD-iPSC line at day 11 of differentiation (midbrainprecursor stage) for FOXA2 (red), LMX1A (green) and DAPI (blue) (A), day25 of differentiation (early postmitotic DA neuronal stage) for FOXA2(red) and TH (green) (B) and for NURR1 (red) and TH (green) (C). D-F)Same set of immunocytochemical analyses performed using H9 derived cellsat day 11 of differentiation for FOXA2 (red), LMX1A (green) and DAPI(blue) (D), at day 25 of differentiation for FOXA2 (red) and TH (green)(E) and for NURR1 (red) and TH (green) (F).

FIG. 21 shows an exemplary PINK1 mutant PD-iPSC showed PD like phenotypeof protein aggregation following long-tenn differentiation andmaturation in vitro. At day 55 of differentiation, PINK1 mutant PD-iPSCshowed evidence of α-synuclein (a major component of Lewy body formationin PD patients) expression in cytosol of TH+DA neurons. The cells alsoshowed high expression of ubiquitin (a classical Lewy body marker). Incontrast, DA neurons derived from control iPS line showed expression ofnormal synaptic (as opposed to cytosolic) α-synuclein expression andvery low levels of ubiquitin. A, B) Immunocytochemical analysis of PINK1mutant PD-iPSC line at day 55 of differentiation for α-synuclein (LB509,red), TH (green) and merged image (A) and α-synuclein (red) andUbiquitin (green) (B). C, D) Immunocytochemical analysis of control-iPSCline at day 55 of differentiation for α-synuclein (red) and TH (green)(C) and α-synuclein (red) and Ubiquitin (green) (D).

FIG. 22 shows an exemplary expression of aggregated form of α-synuclein.In the PD patient brain, dimerized insoluble forms of α-synuclein leadto aggregation in Lewy body. The dimerized form of α-synuclein showsphospholylation of Serine 129 on α-synuclein. PINK1 mutant PD-iPSCderived cells showed strong expression for Ser129 phosphorylatedα-synuclein in contrast to control-iPSC derived cells that showed verylow levels of expression. A, B) Immunocytochemical analysis for Ser129phosphorylated α-synuclein (green) and DAPI (blue) in PINK1. mutantPD-iPSC derived cells at day 55 of differentiation (A) and matchedcontrol-iPSC derived cells (B).

FIG. 23 shows exemplary differences in α-synuclein expression patternsare observed depending of differentiation protocol. The inventors' showthat ‘authentic’ midbrain DA neurons have PD specific vulnerability andcorresponding, specific in vitro phenotypes. DA neurons obtained usingthe classical MS5 stromal feeder based differentiation protocol (Perrieret al., PNAS 2004, herein incorporated by reference) can yield largenumbers of TH+ neurons. However, based on the data of the presentinventions, the TH+ cells resulting from differentiation by theclassical MS5 stromal feeder protocol are not authentic midbrain DAneurons. In cultures differentiated via the MS5 protocol, there weremany α-synuclein positive cells. However, those cells did not co-expressTH. Moreover, there was no difference in expression patterns betweenPD-iPSC and control-iPSC when using the MS5 differentiation strategy.These data indicate that α-synuclein is also expressed in other non-DAcell types and that such non-DA α-synuclein is unchanged in diseaseversus control-iPSC derived cells—particularly when using standard MS5differentiation protocols. Finally, the new floor plate baseddifferentiation protocol of the present inventions yields large numberof TH+ cells co-expressing α-synuclein. Those TH+ cells expressα-synuclein in a cytosolic expression pattern. A, B) Immunocytochemicalanalysis for α-synuclein (LB509, red), TH (green) of PINK1 mutantPD-iPSC line at day 60 of MS5 based differentiation (A) and control-iPSC(B). C) Immunocytochemical analysis of PINK1 mutant PD-iPSC line at day55 of floor-plate based differentiation for α-synuclein (red), TH(green).

FIG. 24 shows an exemplary DA neurons derived from PINK1 mutant PD-iPSCare more vulnerable to toxic stimulation. PD-iPSC derived TH+ DA neuronsderived via the floor-plate based protocols of the present inventionswere more vulnerable to toxin challenge (valinomycin: mitochondriaionophore, SuM, 48 hr) than control-iPSC derived cells. In contrast, TH+neurons derived via the classic MS5 based protocol did not showdifferential vulnerability between PD-versus control-derived cells. A-F)Representative TH immunocytochemistry at day 60 of differentiation:Normal condition (no toxin treatment) for both PD- and control-iPSCderived cells obtained via floor-plate based protocol (A, PD-iPSCderived cells shown), nearly complete degeneration of TH+ DA neurons inPD-iPSC following toxin treatment (B), partially degenerated TH+ DAneurons from control-iPSC (C), Normal condition both of PD- andcontrol-iPSC derived cultures obtained via MS5 based protocol (D,PD-iPSC derived cells shown), TH+ neurons following toxin challenge inPD-iPSC (E), and control-iPSC derived cultures (F) obtained via MS5protocol. G-H) low power images of immunocytochemistry for Tuj1 (red)and TH (green) by floor-plate based protocol at day 60 ofdifferentiation: PD-iPSC of normal (G), versus toxin challenge (H)conditions and control iPSC of normal (I), versus toxin challenge (J)conditions. K-N) low power images of immunocytochemistry for Tuj1 (red)and TH (green) by MS5 based protocol at day 60 of differentiation:PD-iPSC of normal (K), versus toxin challenge (L) conditions and controliPSC of normal (M), versus toxin challenge (N) conditions.

FIG. 25 shows an exemplary quantification of cell viability-doseresponse assay for toxin challenge. Cell viability assay withalamar-blue after 48 hrs of valinomycin treatment showed differentialcell survival in a specific dose range for toxin challenge (5 and 10 uM)when comparing PD-iPSC and control iPSC (day 60 of floor-plate baseddifferentiation of the present inventions). Note: this assay tests foroverall cell death while the most dramatic effects were observedspecifically in DA neurons (see FIG. 14). Therefore, alamar blue basedquantification will likely underestimate the extent of the differentialeffect observed on DA neuron lineages.

FIG. 26 shows exemplary grafted human DA neurons derived frompluripotent stem cells have electrophysiological features typical ofthose seen in mouse substantia nigra pars compacta (SNpc). A)Top-Reconstruction of a pacemaking neuron in the graft region.Bottom-photomicrograph of a brain slice taken from the rat into whichthe hES-derived neurons were injected 9 months prior; the graft isoutlined; a higher magnification image is shown inset at the bottom. Theslice was processed for tyrosine hydroxylase, see white areas. B).Top-cell-attached patch recording from a putative DA neuron in thegraft; Bottom-whole cell recording from the same cell. Recordings weremade in the presence of glutamate and GABA receptor antagonists (50 μMAP5, 10 μM CNQX and 10 μM GABAzine) to eliminate synaptic input. Theserecordings demonstrate that the PS-derived neurons were autonomouspacemakers with normal intrasomatic voltage trajectories. Another neuronrecorded in graft had similar properties. C) For comparison,cell-attached and whole cell recordings from a dopaminergic neuron inSNpc of an adult mouse are shown. Abbreviations (CTx=cortex,STr=striatum, SNpc=substantia nigra pars compacta, DA=dopaminergic).

FIG. 27 shows an exemplary A9 candidate surface marker and CD-screen inhPSCs. a) Venn Diagram of transcriptome data from FACS purified mouseESC derived mDA neurons. Among the 107 genes shared between PITX3 andNurr1 the majority were known markers of midbrain DA neurons as well asnovel markers were confirmed expressed within the ventral midbrain: b)One of those markers was DCSM1, a putative surface marker that appearsto be enriched within the A9 region, based on mRNA in situ expressiondata (Allen Brain Atlas, Lein, E. S. et al., Genome-wide atlas of geneexpression in the adult mouse brain, Nature 445: 168-176 (2007)). c-f)CD screen: c) representative 96 well plate (1 out of 3×96 plates used toscreen complete CD panel). Dark wells label CD markers that are highlyexpressed in hESC DA neurons at day 25. e) Summary of the CD screeningresults in hESC derived DA neurons. f) One exemplary marker, CD142, asurface marker enriching specifically for DA neurons at the Nurr1+ stagewas as found following FACS mediated isolation of CD142+ versus CD142−cells and analysis at day 7 post sort.

FIG. 28 shows exemplary CD142 enriched for Nurr1+ midbrain DA neuronstage and depletes for GABA and Serotonergic neurons. a) Flow cytometryshowed representative CD142 expression on day 25 of differentiation. b)CD142 enriched for Nurr1+ stage among FOXA2/TH midbrain DA neurons inhESCs (e.g. WA09; FIG. 27f ) and hiPSC lines tested (C29, and M3Xrepresent two human iPSC lines at day 25 of differentiation). c, d)CD142 depletes GABAergic and Serotonergic contaminants following day 25sorting and in vitro culture for 3 weeks. e, f) CD142 depletes GABAergicand Serotonergic neurons in vivo 3 months after transplantation. Cellswere sorted for CD142 at day 25. Note: CD142 cells also enriched for THand AADC.

FIG. 29 shows an exemplary contemplated experimental use of PSA.PST-expressing and PSTnm exposed hESC derived DA neurons will beassessed in vitro for impact on DA phenotype and fiber outgrowth. Invivo studies in 6OHDA rat model will be tested for whether lower numbersof DA neurons with forced PSA expression can match behavioral recoveryof standard grafts, and whether forced PSA expression in hESC-derived DAneurons is capable of inducing recover in assays of complex motorfunction.

FIG. 30 shows an exemplary use of PST. Overexpression (mouse PST)resulted in increased levels of PSA-NCAM in differentiating mouse EScells. (A) Quantification of PST mRNA by qPCR in control cells (Nurr1)and in cells overexpressing PST (Nurr1/PST). Data is expressed as thefold enrichment of PST levels in Nurr1/PST versus Nurr1 cells. (B) PSAimmunostaining in DA neuron cultures at day 14 of differentiation showsincreased levels of PSA in Nurr1/PST cells (Scale bar: 100 μm). (C)Western Blot for NCAM in differentiated cells. Nurr1/PST cells (lane 2)shows increased levels of the polysialylated form of NCAM (smear,brackets) compared to control (lane 1). PSA is removed from NCAM afterendoN treatment (lane 3). (D) Quantification of the intensity of the PSAsmear expressed in arbitrary units. (E) PSA FACS analysis at day 14 ofdifferentiation. Treatment of cells with 20 units of endoN, 24 hoursbefore the end of differentiation, abolished the PST effect. (F)Representative photomicrographs comparing Nurr1 and Nurr1/PSTdifferentiated cells for GFP immunofluorescence and DA markers. Cellssorted for GFP and re-plated still retained the DA phenotype (postsort). Scale bars: 100 μm.

FIG. 31 shows an exemplary FACS analysis of ES-derived DA neurons. Flowcytometry-based isolation of GFP+ and SSEA-1-cells. As double negativeand GFP negative controls, J1 mouse ES-cells were used. Around 5 to 10%of cells were sorted positively.

FIG. 32 shows exemplary Nurr1/PST grafts were more effective at inducingbehavioral recovery in 6OHDA mouse model. Nurr1::GFP cells weredifferentiated and sorted at day 14 for GFP+/SSEA-1-population. Cellstreated with endoN were cultured for 12 hours before sorting with 20units of the enzyme. 55,000 cells were grafted in 1 ml of N2 media withBDNF and AA. (A) Animals scored for amphetamine-induced rotation(rotations/min during 20 min) for 3 weeks prior to grafting, then for 7weeks after. Nurr1/PST cells significantly improve the outcome ascompared to Nurr1 controls (2-way-ANOVA: p<0.01, with Bonferronipost-test: *p<0.05, **p<0.01, ***p<0.001; 6 animals/group). Removal ofPSA by endoN abolishes the PST effect (p=0.26). (B) There were more GFP+cells in the PST graft at endpoint than in control (p<0.05, t-test). (C)Ratio of PSA/GFP immunofluorescence at the core. **p<0.01. Student's ttest (n=5/graft type). (D) GFP, TH and PSA immunofluorescence. Scalebar: 200 μm. (E) Grafted cells express DA markers. Individual z-planesof confocal micrographs are shown. Scale bar: 20 μm. Values aremeans+/−SEM.

FIG. 33 shows exemplary PSA augmentation that increased host striatuminnervation by ES-derived DA neurons. PSA-NCAM overexpression increasedprocess outgrowth. (A) Representative photomicrographs of GFP/TH+,GFP/Girk2+ and GFP/synapsin+ processes in controls. Staining inNurr1/PST samples was similar. Scale bar: 20 μm. (B) Representativez-stack projections showing GFP+ processes extending out of both Nurr1and Nurr1/PST grafts. There are more GFP+ processes extending out of theNurr1/PST graft (scale bar: 50 μm). Insets show GFP+/TH+ processes insame sections. Arrow: direction of growth. (C-E) Quantification of theintensity of GFP+ (C, E) and TH+(D) processes at different distancesfrom the graft site normalized to the intensity nearest to the injectionsite (areas were divided into five zones of ˜100 μm apart. Normalizationis relative to zone I; Two-way ANOVA, p<0.01 for both GFP and TH data,n=5/cell type; values are means+/−SEM). Nurr1/PST grafts grew moreneurites into host striatum, which was partly suppressed (E) by endoNtreatment. (F) Animal recovery correlated with the degree of processoutgrowth. The graph shows the correlation (linear regression analyses)between the intensity of GFP+ neurites in zone IV and animal recoveryfor untreated and endoN-treated Nurr1 and Nurr1/PST grafted animals.Each value represents one animal (r 2=0.65, p<0.001, n=17).

FIG. 34 shows an exemplary use of PST in methods associated with spinalcord injury and for expression on motoneurons. In control spinal cord,grafted GFP Schwann cells (SCs) are restricted to the lesion site byhost scar tissue (A), whereas PST-modified SCs readily migrateconsiderable distances (B). This PSA engineering resulted in improvementin locomotion, BBB subscore; upper line in (C) shown vs lower linecontrol) and hindlimb dexterity (gridwalk test; lower line in (D) vsupper line controls). E, H): differentiation of HB9::GFP mouse ESCs intomotoneurons in which PST introduction increases fiber sprouting and cellmigration (arrowheads) in vitro (E, F). Grafting of these PST-cells intosciatic nerve results in better target innervation as shown by thenumbers of neuromuscular junctions (arrows) in the EDL muscle (G, H).

FIG. 35 shows an exemplary use of PSTnm enzyme. (A) PSTnm-produced PSAinhibits adhesion of Schwann cells in suspension to a Schwann cellmonolayer even more effectively (red line-lowest line) than PSA producedby forced PST expression (green line-middle line). (B) PSAimmunoblotting in ESC-derived 11B9 motoneurons shows that controlsamples treated with PSTnm have undetectable levels of PSA. Incubationwith PSTnm+CMP-sialic acid substrate produces a large PSA band, which isremoved with endoN treatment. (C, D) Similar to effects obtained withthe PST gene, polysialylation of these cells by PSTnm and substrateduring differentiation enhances neurite outgrowth and cell migration(arrowheads). (E) PSA immunostaining of day-30 hESC-derived DA neurons.(F) This staining is significantly increased after treatment with PSTnmand substrate. (G) In vivo injection of PSTnm alone has no effect, whileits co-administration with substrate (H) produces large amounts of PSAexpression in mouse striatum.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of stem cell biology, inparticular the linage specific differentiation of pluripotent ormultipotent stem cells, which can include, but is not limited to, humanembryonic stem cells (hESC) in addition to nonembryonic human inducedpluripotent stem cells (hiPSC), somatic stem cells, stem cells frompatients with a disease, or any other cell capable of lineage specificdifferentiation. Specifically described are methods to direct thelineage specific differentiation of hESC and/or hiPSC into floor platemidbrain progenitor cells and then further into large populations ofmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons using novel cultureconditions. The midbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons madeusing the methods of the present invention are further contemplated forvarious uses including, but not limited to, use in in vitro drugdiscovery assays, neurology research, and as a therapeutic to reversedisease of, or damage to, a lack of dopamine neurons in a patient.Further, compositions and methods are provided for differentiatingmidbrain fate FOXA2+LMX1A+TH+ dopamine (DA) neurons from humanpluripotent stem cells for use in disease modeling, in particularParkinson's disease.

The present inventions relate to characteristics of Parkinson's disease(PD) including the selective degeneration of midbrain dopamine (mDA)neurons in patients' brains. Because PD symptoms are primarily due tothe selective loss of DA neurons in the substantia nigra of the ventralmidbrain, PD is considered one of the diseases most suitable for cellreplacement therapeutic strategies for treatment. Thus many attemptswere made to transplant cells into patients' brains in order to replacethe loss of function of the midbrain dopamine (mDA) neurons. Howeverthese experiments were unsuccessful and currently symptomatic treatmentsused on patients have a wide variability of success. Therefore, newtreatments are needed for patients with PD in order to slow the loss ofneuronal function.

Human pluripotent stem cells (hPSCs) are a source of cells forapplications in regenerative medicine. Directed differentiation of hPSCsinto specialized cells such as spinal motoneurons (Li, et al. Nat.Biotechnol. 23, 215-221 (2005), herein incorporated by reference) ormidbrain dopamine (DA) like neurons resulting from differentiation bymethods other then the methods of the present invention. The inventors'discovered as described herein that previous dopamine (DA) neurons (i.e.In Perrier, et al Proc Natl Acad Sci USA 101, 12543-8 (2004), hereinincorporated by reference) referred to herein as dopamine (DA)-likeneurons are not authentic midbrain dopamine (DA) neurons of the presentinventions (see FIGS. 3, 10, 13 and 16). Therefore, the inventorslabeled the floor-plate derived dopamine producing neurons made bymethods described herein, i.e. dopamine producing neurons of the presentinventions as “authentic” because unlike dopamine producing neurons madeby published methods, when “authentic” dopamine producing neurons of thepresent inventions are transplanted into rodents and primates theyreverse Parkinson-like neurological conditions with less interferencefrom neural overgrowth and teratoma formation. Also, unlike previousmethods of making dopamine producing neurons, “authentic” dopamineproducing neurons of the present inventions are produced at higherpercentages from starting populations and retain engrafting capabilityfor several months in culture.

Thus, methods for making authentic midbrain DA neurons were discoveredby using the differentiation methods described herein. However, theeffective use of hPSCs for cell therapy has lagged far behind cellculture advances. While mouse PSC-derived DA neurons have shown efficacyin models of Parkinson's Disease (PD) (Tabar, et al. Nature Med. 14,379-381 (2008); Wernig, et al. Proc. Natl. Acad. Sci. U. S. A 105,5856-5861 (2008), all of which are herein incorporated by reference), DAneurons derived from human PSCs generally display poor in vivoperformance (Lindvall and Kokaia, J. Clin. Invest 120, 29-40 (2010),herein incorporated by reference). In addition to not compensating forthe endogenous loss of neuronal function, there are serious safetyconcerns when hPSC derived neurons are used for transplantation and arerelated to their potential for teratoma formation or neural overgrowth(Roy, et al. Nature Med. 12, 1259-1268 (2006); Elkabetz, et al. GenesDev. 22, 152-165 (2008), herein incorporated by reference).

Another possible source of cells for transplantation are DA neuronsderived from human ESCs. Previous attempts using these cells as astarting cell population to make differentiated cells that appeared tobe DA like-midbrain neurons derived from human embryonic stem cells(hESCs) that were transplanted into rodent PD models resulted in poor invivo survival of the transplants after transplantation. This failure wascontemplated to most likely be due to incomplete midbrain DA neurondifferentiation in vitro resulting in cells that appeared to be midbrainDA neurons but were not capable of engraftment to replace lost neuronfunction. In fact, the inventors show herein that DA-like neuronspreviously made in their laboratories and described in publications werenot the same cell type nor had similar functions or engraftmentcapabilities as the floor-plate midbrain DA neuronal cells of thepresent inventions, see, FIGS. 16 and 17 for examples. Therefore theinventors also discovered that in order for cells to undergo directeddifferentiation in the laboratory to produce cell populations containinglarge numbers of properly functioning neurons, the cells needed to gothrough the specific developmental stages in order to become a suitablereplacement cell population for cell based replacement therapies. Theinventors also discovered that, at least for obtaining the engraftableDA neurons of the present inventions, certain developmental stages mustbe present, such as the FOX2A/LIM1A+ Day 11 intermediates. If suchdevelopmental stages are not present, the inventors' discovered thatresulting DA-like neurons do not have the same functional capabilitiesas the midbrain DA neurons of the present inventions that were derivedfrom FOX2A/LIM1A+ Day 11 intermediates.

In contrast to previous observations, novel culture techniques relatedto floor plate cell induction-based strategies for the derivation ofhuman DA neurons that efficiently engraft in vivo are described herein.Thus past failures in obtaining cell populations comprising primarilycommitted DA neurons of the present inventions (i.e. FOXA2+ and LMX1A+DAneurons capable of efficient engrafting) were contemplated to be thereason that engraftment of the DA like neurons failed, i.e. due toincomplete specification of the DA like cells. Previous hypothesis werethat transplant failure was due to specific vulnerability of the cells,i.e. DA like cultured neurons were unable to survive the stress oftransplantation. As described herein, midbrain FOXA2+/LMX1A+ floor plateprecursors were derived from hPSCs in 11 days following exposure tosmall molecule activators of sonic hedgehog (SHH) and canonical WNTsignaling. These Day 11 cells, double positive for FOXA2+ and LMX1A+,are contacted with additional small molecules to induce furtherdifferentiation into engraftable midbrain DA neurons, positive forTH+FOXA2+ and LMX1A+, by day 25. These mature floor-plate midbrain DAneurons can be maintained in vitro for several months. Extensive invitro molecular profiling, biochemical and electrophysiological datadefined developmental progression and confirmed identity of hPSC-derivedmidbrain DA neurons. In vivo survival and function was demonstrated inPD animal models in three host species. Long-term engraftment in6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrainDA neurons, complete restoration of amphetamine-induced rotationbehavior and improvements in tests of forelimb use and akinesia.Finally, scalability is demonstrated by transplantation intoParkinsonian monkeys. Excellent DA neuron survival, function and lack ofneural overgrowth in the three animal models tested indicate promise forthe development of cell based therapies in PD based on the compositionsand methods of the present inventions.

Therefore the inventors' contemplate the main use of their discoveriesas the capability to produce an unlimited supply of fully functionalfloor-plate derived midbrain DA neurons suitable for pre-clinical andclinical therapeutic applications. Specifically, the inventors'discovered a new protocol for the efficient differentiation of mDAneurons from at least pluripotent cell populations isolated from rodentsand humans (human embryonic stem cell (hESC) and human inducedpluripotent stem cells (hiPSCs)). Those studies included PINK1 mutantiPS cell lines derived from a human patient suffering from a geneticform of Parkinson's disease Seibler, et al., The Journal ofNeuroscience, 2011, 31(16):5970-5976, herein incorporated by reference.Human stem cells populations (hESC or hiPSC) were differentiated into amidbrain phenotype, which after contacting with neuronal maturationmolecules gave rise to more authentic engraftable DA neurons. Thisprotocol was used to demonstrate high yields of hESC progeny by Day 11of directed differentiation into a midbrain DA (mDA) neuronal phenotypewhich included expression of key transcription factors e.g TH, FoxA2 andLMX1A which upon further differentiation yielded additional key proteinse.g. TH. Transplantation of these hESC derived mDA neurons intoimmunocompromised rodent and primate hosts, unlike previous in vitroderived DA neurons, showed good in vivo survival of the grafted cellswith functional restoration of behavioral deficits.

Advantages of using methods of the present inventions for producing DAneuronal cells over other methods are, in part, evident from thefollowing information. The use of somatic stein cells and neural stemcells in other methods with the goal of generating authentic midbrain DAneurons that efficiently engraft in vivo have not been successful (forreview see Kriks & Studer, Protocols for generating ES cell-deriveddopamine neurons in Development and engineering of dopamine neurons(eds. Pasterkamp, et al.) (Landes Biosciences, 2008, herein incorporatedby reference). Pluripotent stem cells such as ES cells were then used assources for generating engraftable cells. Early studies in the 1990susing mouse ES cells demonstrated the feasibility of deriving specificlineages from pluripotent cells in vitro including neurons (Okabe, etal., Mech. Dev. 59:89-102 (1996); Bain, et al., Dev. Biol. 168v342-357(1995), all of which are herein incorporated by reference). In fact,midbrain DA neurons were generated using a directed differentiationstrategy (Lee, et al., Nat. Biotechnol. 18v675-679 (2000), hereinincorporated by reference) based on developmental insights from earlyexplants studies (Ye, et al., Cell 93:755-766 (1998), hereinincorporated by reference). Other directed differentiation strategieswere used for producing other neuron types such as somatic motoneurons(Wichterle, et al., Cell 110, 385-397 (2002), herein incorporated byreference). However, these efforts did not result in cell populationscontaining high percentages of midbrain DA neurons or cells capable ofrestoring neuronal function in vivo. In fact, the resulting populationcontained a mixture of cell types in addition to midbrain DA neurons.Even the inventors developed other methods of making midbrain DAneurons, in part, see below, however these cell populations were alsomixtures of cell types and failed to restore neuronal function. Inparticular, human ES cells were differentiated into earlyneuroepithelial cells, termed neural rosettes, followed by induction ofrosette stage cells into cells expressing midbrain DA neuron precursorand differentiated markers. Those cells went on to exhibit functionalneuronal features by electrophysiology, in vitro DA release and theformation of TH-immunogold positive synaptic contacts (Perrier, et al.From the Cover: Derivation of midbrain dopamine neurons from humanembryonic stem cells. Proc Natl Acad Sci USA 101, 12543-8 (2004), hereinincorporated by reference). However, despite these promising in vitrodata, transplantation of the cells in 6OHDA lesioned murine hostsresulted in a very small number of surviving dopaminergic neurons. Thiswas a surprising negative result given strong evidence of in vivofunctionality of mouse ES derived DA neurons (Barberi, et al., Nat.Biotechnol. 21:1200-1207 (2003); Tabar, et al. Nature Med. 14:379-381(2008); Bjorklund, et al. Proc. Natl. Acad. Sci. U. S A. 99:2344-2349(2002); Kim, et al. Nature 418:50-56 (2002), all of which are hereinincorporated by reference), robust in vitro functional features of humanES derived DA neurons (Perrier, et al., From the Cover: Derivation ofmidbrain dopamine neurons from human embryonic stem cells. Proc NatlAcad Sci USA 101:12543-8 (2004), herein incorporated by reference) andclear evidence that human fetal DA neurons can survive as striatalxenografts (Brundin, et al. Exp. Brain Res. 70:192-208 (1988);Bjorklund, et al., Neuronal replacement by intracerebral neural implantsin animal models of neurodegenerative disease. Raven. Press., New. York.455-492 (1988), all of which are herein incorporated by reference). Fornearly 8 years after initial failed attempts of grafting human ES cellderived DA neurons there was very little progress in the field. Somelimited improvements were observed using primate pluripotent stem cellsources (Sanchez-Pernaute, et al. Long-term survival of dopamine neuronsderived from parthenogenetic primate embryonic stem cells (Cynol) in ratand primate striatum. Stem Cells 23:914-922 (2005), herein incorporatedby reference), cells pretreated with FGF20 or Wnt5A or human ES cellsdifferentiated in the presence of factors secreted from immortalizedmidbrain astrocytes (Roy, et al., Nature Med. 12:1259-1268 (2006),herein incorporated by reference). However, none of the previousstrategies were success in producing an enriched population of DAneurons of the present inventions for use in engraftment procedures forrestoring neuronal function in vivo.

I. Cell Culturing Methods for Inducing Neuronal Precursor (Lineage)Cells: Contacting Human Pluripotent Stem Cells With SB431542 andLDN-193189 Resulted In Differentiated Neural Lineage Cells.

The following example describes exemplary methods for providing cells ofa neural lineage for use during development of the present inventions.

Dual SMAD inhibition was previously used as a rapid and highly effectivemethod for inducing one type of neural lineage cells from hPSCs(Chambers, et al., Nat Biotechnol 27, (2009), herein incorporated byreference). These neural lineage cells induced by molecules, includingNoggin, had a default pathway that allowed development into centralnervous system cells, i.e. neural cell fate. Follow up studies reportedthe use of a small molecule dorsomorphin (DM) instead of Noggin that, atleast in part, resulted in similar but not the same differentiated cellswith major differences in consistency of cultures (Kim, et al., Robustenhancement of neural differentiation from human ES and iPS cellsregardless of their innate difference in differentiation propensity.Stem Cell Rev 6, 270-281, (2010); Zhou, et al., High-EfficiencyInduction of Neural Conversion in hESCs and hiPSCs with a SingleChemical Inhibitor of TGF-beta Superfamily Receptors. Stem Cells, 504,(2010), herein incorporated by reference).

The inventors observed that cells generated using Noggin, despiteshowing the same developmental stage as LDN treated cells, express thevast majority of the same markers, and are capable of a similardevelopmental potential to make various neural lineages, but also showeddifferences, such as being more anterior on an anterior-posterior axis(i.e. more forebrain, more cells express FOXG1, and the like) comparedto neural cells induced using LDN. Thus, although LDN was used in placeof Noggin to inhibit BMP among other signaling pathways, Noggin and LDNmay have other types of activities which are different than inhibitionof BMP.

In part due to the high expense of using Noggin, the inventorscontemplated that the use of a BMP inhibitor might be able to substitutefor Noggin in differentiating cells of neural cell fate. Therefore, asmall molecule BMP inhibitor, LDN-193189, (Yu, et al., Nat Med 14,1363-1369, (2008), herein incorporated by reference) was used and foundduring the development of the present inventions to replace Noggin, incombination with SB431542, for generating primitive neuroectoderm fromhPSCs, cells that have neural cell fate, i.e. CNS cells (FIG. 2A). Thiscombination treatment was termed LSB for the combination of these twoinhibitors LDN-193189 and SB431542.

In general, cell differentiation was initiated by treatment of highconfluency monolayer hES or hiPS with dual inhibition of SMAD signaling.A preferred embodiment utilizes a percentage confluency of 50%400%, witha most preferred embodiment of 70%-80% conflucncy. It will be obvious toone skilled in the art that the initial plating density required toachieve a preferred confluency of the present invention will bedependent on cell type, size, plating efficiency, survival, adhesion andother parameters which can be determined empirically without undueexperimentation on the part of the skilled artisan. Dual inhibition ofSMAD can be achieved with a variety of compounds including Noggin,SB431542, LDN-193189, Dorsomorphin, or other molecules that block TGFβ,BMP, and Activin/Nodal signaling. A preferred embodiment utilizes thecomposition comprising SB431542 and LDN-193189 (collectively, LSB) at aconcentration of 0.1 μM-250 μM, or more preferable 1-25 μM, or mostpreferable 10 μM of SB431542 and 10-5000 nM, or most preferably 100-500nM of LDN-193189.

II. Derivation of DA Neurons from hESCs Through Rosette CellIntermediates and Results of Transplant Studies that Used these DA-LikeNeurons.

The inventors previous used several other directed differentiationmethods that resulted in cell populations containing DA-like neurons.These DA-like neurons were used in transplantation studies that resultedin concerns on the further use of these cells for therapeuticapplications. For examples, procedures described in Perrier et al., 2004and Fasano et al., 2010, including MS5 neural induction, resulted inrosette cell formation and were used to make Day 11 precursors, seeFIGS. 2, 16 and 17 for examples, that were further used to deriveDA-like neurons. These neurons resulted from a low percentage of theprecursor cells in the resulting Day 11 cell populations.Transplantation studies that used these neurons showed poor posttransplant viability and loss of the DA-like neuronal phenotype inaddition to observations of post transplantation development ofinappropriate neural types along with loss of growth control, which ledto development of teratomas. See FIGS. 16 and 17.

Specifically, at P0 hESCs were contacted with molecules for beginningneural induction of Oct4+ cells into rosette cells using MS5 feedercells (Perrier et al., 2004). At the P1 stage Rosette cells wereexpanded by contacting cells with additional molecules fordifferentiating cells into cells at stage P2 with specific expressionpatterns including Pax2+/En1+ DA progenitor cells and were furtherdifferentiated into TH+/En1+ DA neurons. These cells were used forengraftment in 6OHDA lesioned rats, immunosuppressed with Cyclosporin A.Those transplantation studies showed poor in vivo viability, loss of theTH+ phenotype, concerns about further growth into unwanted, possiblylethal, cells, i.e. teratomas, and growth of cells into inappropriateneural types that would cause additional medical problems for thepatient.

There were very small numbers of surviving TH+ neuron at 4.5 moths aftertransplantation (<50 TH+ cells/animal) in grafts from rosette derived DAneuron precursors FIG. 16A. However, in contrast to TH+ cells, GFPmarked cells (GFP was driven by a ubiquitous promomoter) did survivequite well after transplantation. This suggests that most survivingcells following transplantation were neural cells of non-DA neuronidentity (16B). Few graft-derived cells (hNA+(green) co-express TH (red)again suggesting that most grafted human cells adopt a non-DA neuronphenotype FIG. 16C. Panels 16 D-E show that D-E, despite the very poorin vivo survival there was some (low and highly variable) improvement ina few behavioral assays such as amphetamine induced rotations (D),cylinder test and spontaneous rotations (E). Feeder-free neuralinduction was carried out as previously described (Chambers et al.,2009, herein incorporated by reference) but further modified to yieldfloor plate cells (Fasano et al., 2010, herein incorporated byreference).

In the modified Dual-SMAD inhibition method for differentiatingpluripotent cells into floor plate cells, the inventors' previouslydiscovered that high concentrations of SHH were required for FPinduction by day 11. For example, in some embodiments, Sonic C25II wasadded at 200 ng/ml. In some experiments, DKK-1 (R&D; 100 ng/ml) FGF8(R&D; 50 ng/ml), Wnt-1 (Peprotech; 50 ng/ml) and retinoic acid (R&D; 1mM) were added, See FIG. 17. However none of the resulting cellpopulations at day 11 using previous methods, contained the highpercentage of FOXA2+/LMX1A+ midbrain floor-plate progenitor cells usingmethods of the present inventions.

III. Compounds for Use in Directed Differentiation: Screening SmallMolecules Using Neuronal Lineage Cells of the Present InventionsResulted in Compounds that Differentiated Cells into FOX2A+ and LIMX1A+Neuronal Cells by Day H of Culture.

The following example describes using exemplary cells from Section I forscreening small molecule candidate compounds and determining whethertheir use would result in directed differentiation of a cell populationcontaining a high percentage of midbrain floor plate neurons by Day 11after the initial contact with the Dual-SMAD inhibitors. The results ofthis screen initially showed that a SHH activating molecule togetherwith activation of FGF8 and Wnt led to the efficient derivation ofFOXA2+/LMX1A+ positive midbrain floor plate cells from hESC by day 11 ofdifferentiation. The inventors show results herein of exemplaryexperiments that defined which molecules were necessary and the optimaltime of contacting in order to derive the desired FOXA2+/LMX1A+ positivecell population at Day 11.

Recent mouse genetic studies demonstrated an important role for thetranscription factor FOXA2 in midbrain DA neuron development andsurvival. A unique feature of the developing midbrain is theco-expression of the floor plate marker FOXA2 and the roof plate markerLMX1A. Normally, floor plate and roof plate cells are located atdistinct positions in the CNS (ventral versus dorsal) with diametricallyopposed patterning requirements for their induction. Derivation ofregion-specific floor plate precursors from hESCs using a modifiedDual-SMAD inhibition protocol was recently described. Canonical Wntsignaling was important for both roof plate function and midbrain DAneuron development.

A. CHIR99021 (CHIR) induced a high yield of midbrain DA neuron precursorfate by Day 11 of culture. As described herein, exposure to CHIR99021(CHIR), a potent GSK3β inhibitor known to strongly activate WNTsignaling, induced LMX1A in FOXA2+ floor plate precursors (FIG. 1a ).CHIR was more potent than recombinant Wnt3A or Wnt1 at inducing LMX1Aexpression. The efficiency of LMX1A induction was dependent on thetiming of CHIR exposure with a maximum effect at day 3-11 (FIG. 5). CHIRinduced co-expression of FOXA2/LMX1A, while other factors such as FGF8had only marginal effects (FIG. 6). Induction of FOXA2/LMX1Aco-expression required strong activation of SHH signaing usingpurmorphamine, a small molecule agonist, alone or in combination withrecombinant SHH (FIG. 7).

Treatment with SHH agonists (purmorphamine+SHH) and FGF8 (S/F8) in theabsence of CHIR99021 showed significantly lower expression of FOXA2 byday 11 and complete lack of LMX1A expression (FIG. 1a,b ). Dual-SMADinhibition (exposure to LDN-193189+SB431542=“LSB”) did not yieldFOXA2-expressing cells, but did yield a subset of LMX1A+ cells (FIG.1a,b ). The anterior marker OTX2 was robustly induced in LSB andLSB/S/F8/CHIR treated cultures, but not under LSB/S/F8 conditions (FIG.1a,c ). Systematic comparisons of the three culture conditions (FIG. 1d) were performed using global temporal gene expression profiling.Hierarchical clustering of differentially expressed genes segregated thethree treatment conditions by day 11 of differentiation (FIG. 8a ).FOXA1, FOXA2 and several other SHH downstream targets including PTCH1were amongst the most differentially regulated transcripts inLSB/S/F8/CHIR versus LSB treatment sets (FIG. 1e ). Expression of LMXIA,NGN2, and DDC indicated establishment of midbrain DA neuron precursorfate already by day 11 (FIG. 1e,f ). In contrast, LSB cultures wereenriched for dorsal forebrain precursor markers such as HES5, PAX6,LHX2, and EMX2. Direct comparison of LSB/S/F8/CHIR versus LSB/S/F8treatment (FIG. 1f ) confirmed selective enrichment for midbrain DAprecursor markers in LSB/S/F8/CHIR group and suggested hypothalamicprecursor identity in LSB/S/F8 treated cultures based on thedifferential expression of RAX1, SIX3, and SLX6 (see also POMC, OTPexpression in FIG. 2d below). The full list of differentially expressedtranscripts Tables 1, 2 and gene ontology analysis FIG. 8b (DAVID;http://david.abcc.ncifcrf.gov) confirmed enrichment for canonical WNTsignaling upon CHIR treatment. Raw data are not yet available at GEOworldwideweb.ncbi.nlm.nih.gov/geo/accession#: [TBD]). Comparativetemporal analysis of gene expression for midbrain DA precursor markers(FIG. 1g ) versus markers of anterior and ventral non-DA neuron fates(FIG. 1h ) partitioned the three induction conditions into: 1) LSB:dorsal forebrain; ii) LSB/S/F8: ventral/hypothalamic and iii)LSB/S/F8/CHIR: midbrain DA precursor identity.

Cells resulting from the protocol derived during the development of thepresent inventions were compared to cells derived by previous methods.See, FIGS. 2 and 18 for an exemplary visual comparison to theSHH/FGF8+CHIR treated cells of the present inventions. FIG. 19A showsexamples of FOXA2/Tuj1 double labeled cells following LSB/S/F8/CHIRtreatment (upper panels) and FOXA2 co-labeling with TH, Nurr1 and LMX1A(lower panels). Those marker combinations are diagnostic for early stagemidbrain DA neuron precursors. FIG. 19 B shows gene expression data (forcomparison to FIG. 2E) for key dopamine neuron precursor markers.

In general Materials And Methods Used Herein Are Described: Human ESC(H9, H1) and iPSC lines (2C6 and SeV6) were subjected to a modified DualSMAD-inhibition (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009),herein incorporated by reference) based floor plate induction (Fasano,et al., Cell Stem Cell 6:336-347 (2010), herein incorporated byreference) protocol. Exposure to SHH C25II, Purmorphamine, FGF8 andCHIR99021 were optimized for midbrain floor plate and yield of novelpopulations of DA neuron (see FIG. 1d ). Following floor plateinduction, further maturation (days 11-25 or longer than 25 days inculture up to at least 100 days in culture) was carried out indifferentiation medium based on Neurobasal/B27 in the presence of DAneuron survival and maturation factors (Perrier, et al. Proc Natl AcadSci USA 101:12543-8 (2004), herein incorporated by reference) such asAA, BDNF, GDNF, TGFβ3 and dbcAMP (see full methods for details). Theresulting DA neuron populations were subjected to extensive phenotypiccharacterization via immunocytochemistry, qRT-PCR, global geneexpression profiling, HPLC analysis for the detection of dopamine and invitro electrophysiological recordings. In vivo studies were performed inhemiparkinsonian rodents (mouse or rats injected with the 6OHDA toxin onone side of the animal's brain. The studies were carried out in adultNOD-SCID IL2Rgc mice (Jackson labs) and adult Sprague Dawley ratsTaconic Farms, that received 6-hydroxydopamine lesions by stereotacticinjections of the toxin as described previously as well as two adultrhesus monkeys that were treated with unilateral carotid injections ofMPTP.

DA neurons were injected stereotactically in the striata of the animals(150×10³ cells in mice, 250×10³ cells in rats) and a total of 7.5×10⁶cells (distributed in 6 tracts; 3 on each side of brain) in monkeys.Behavioral assays were performed at monthly intervals post-grafting,including amphetamine mediated rotational analysis as well as a test forfocal akinesia (“stepping test”) and limb use (cylinder test). Rats andmice were sacrificed at 18-20 weeks and the primates at 1 month postgrafting. Characterization of the grafts was performed via stereologicalanalyses of cell number and graft volumes as well as a comprehensivephenotypic characterization via immunohistochemistry.

Culture of undifferentiated human ES cells. hESC lines H9 (WA-09, XX,passages 27-55 from when 10/2009), H1 (WA-01, XY, passages 30-40 fromwhen 6/2010) and iPS cell lines 2C6 (Kim, et al. Cell Stem Cell8:695-706 (2011), herein incorporated by reference) (XY, passages 20-30)and SeV6 (XY, passages 20-30; derived from MRC-5 embryonic fibroblastsusing non-integrating 4 factor Sendai vector system (Ban, et al. Proc.Natl. Acad. Sci. U. S. A (2011)108(34):14234-14239:10.1073/pnas.1103509108, herein incorporated byreference) were maintained on mouse embryonic fibroblasts at platingconcentrations estimated ranging from 0.5×10³ per cm² to 100×10³ per cm²based upon human ES cells which tend to cell cluster. (MEF, Global Stem,Rockville, Md.) in an optimal 20% knockout serum replacement (KSR,Invitrogen, Carlsbad, Calif.)-containing human ES cell medium (asdescribed previously (Kim, et al. Cell Stem Cell 8:695-706 (2011),herein incorporated by reference). The use of knockout serum replacementmay range from 0% to 40%.Neural Induction. For floor plate-based midbrain dopamine neuroninduction, a modified version of the dual-SMAD inhibition (Chambers, etal. Nat. Biotechnol. 27:275-280 (2009), herein incorporated byreference) and floor plate induction (Fasano, et al. Cell Stem Cell6:336-347 (2010), herein incorporated by reference) protocol was usedbased on timed exposure to LDN-193189 (100 nM (ranging in concentrationfrom 0.5-50 μM, Stemgent, Cambridge, Mass.), SB431542 (10 μM (ranging inconcentration from 0.5-50 μM, Tocris, Ellisville, Mich.), SHH C25II (100ng/ml (ranging in concentration from 10-2000 ng/ml, R&D, Minneapolis,Minn.), Purmorphamine (2 μM (ranging in concentration from 10-500 ng/ml,Stemgent), FGF8 (100 ng/ml (ranging in concentration from 10-500 ng/ml,R&D) and CHIR99021 (CHIR; 3 μM (ranging in concentration from 0.1-10 μM,Stemgent).

For the floor plate induction protocol “SHH” treatment refers toexposure, i.e. contact, of cells to a combination of SHH C25II 100ng/ml+Purmorphamine (2 μM). Cells were plated (35-40×10³ cells/cm²) andcultured for 11 days on matrigel or geltrex (used as purchased) (BD,Franklin Lakes, N.J.) in Knockout serum replacement medium (KSR)containing DMEM, 15% knockout serum replacement, 2 mM L-glutamine and10-μM (ranging in concentration from 1-25 μM β-mercaptoethanol. KSRmedium was gradually shifted to N2 medium starting on day 5 ofdifferentiation, by mixing in ratios of 75% (KSR):25% (N2) on day 5-6,50% (KSR):50% (N2) day 7-8 and 25% (KSR):75% (N2) on day 9-10, asdescribed previously (Chambers, et al. Nat. Biotechnol. 27:275-280(2009), herein incorporated by reference). On day 11, media was changedto Neurobasal medium/B27medium (1:50 dilution)/L-Glut (effective ranges0.2-2 mM)) containing medium (NB/B27; Invitrogen) supplemented with CHIR(until day 13) and with BDNF (brain-derived neurotrophic factor, 20ng/ml ranging from 5 to 100; R&D), ascorbic acid (AA; 0.2 mM (ranging inconcentration from 0.01-1 mM), Sigma, St Louis, Mich.), GDNF (glial cellline-derived neurotrophic factor, 20 ng/ml (ranging in concentrationfrom 1-200 ng/ml); R&D), TGF β3 (transforming growth factor type β3,Ing/ml (ranging in concentration from 0.1-25 ng/ml); R&D), dibutyrylcAMP (0.5 mM (ranging in concentration from 0.05-2 mM); Sigma), and DAPT(10 nM (ranging in concentration from 0.5-50 nM); Tocris,) for 9 days.On day 20, cells were dissociated using Accutase® (Innovative CellTechnology, San Diego, Calif.) and replated under high cell densityconditions (for example from 300-400 k cells/cm²) on dishes pre-coatedwith polyornithine (PO); 15m/ml (ranging in concentration from 1-50μg/ml)/Laminin (1 μg/ml) (ranging in concentration from 0.1-10μg/ml)/Fibronectin (2 μg/ml (ranging in concentration from 0.1-20 μg/ml)in differentiation medium (NB/B27+BDNF, AA, GDNF, dbcAMP (ranging inconcentration as described herein), TGFβ3 and DAPT (ranging inconcentration as described herein) until the desired maturation stagefor a given experiment.

For rosette-based DA neuron induction previously described protocolswere followed in part (Perrier, et al. Proc Natl Acad Sci USA101:12543-8 (2004), herein incoropoated by reference) with at least oneexception where dual-SMAD inhibition was used to accelerate the initialneural induction step. In brief, hESCs were induced towards neural fateby coculture with irradiated MS5 cells in KSR supplemented with SB431542and Noggin (250 ng/ml (ranging in concentration from 10-1000 ng/ml);R&D), from day 2-8 and SHH+FGF8 from day 6-11 of differentiation. After11 days in KSR, neural rosettes were manually isolated and cultured (P1stage) in N2 medium supplemented with SHH, FGF8, BDNF and AA asdescribed previously (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8(2004), herein incorporated by reference). After 5-7 days in P1 stage,rosettes were again harvested mechanically and triturated followingincubation in Ca²/Mg²-free Hanks' balanced salt solution (HBSS) for 1 hand replated on polyornithine (PO)/Laminin/Fibronectin coated plates.Patterning with SHH/FGF8 was continued for 7 days at P2 stage followedby final differentiation in the presence of BDNF, AA, GDNF, TGFb3 anddbcAMP as described above until the desired maturation stage for a givenexperiment (typically 5-7 days for transplantation studies or 32 daysfor in vitro functional studies).

Gene expression analyses. Total RNA was extracted during differentiationat days: 0, 1, 3, 5, 7, 9, 11, 13 and 25 from each condition of controlLSB, LSB/SHH/FGF8 and LSB/SHH/FGF8/CHIR using the RNeasy kit (Qiagen,Valencia, Calif.). For microarray analysis, total RNA was processed bythe MSKCC Genomic core facility and hybridized on Illumina Human ref-12bead arrays according to the specifications of the manufacturer.Comparisons were performed among each days and conditions using theLIMMA package from Bioconductor (worldwideweb.bioconductor.org). Genesfound to have an adjusted P-value <0.05 and a fold change greater thantwo were considered significant. Some of the descriptive microarray dataanalyses and presentation was performed using a commercially availablesoftware package (Partek Genomics Suite (version 6.10.0915)). ForqRT-PCR analyses, total RNA at day 25 of each condition was reversetranscribed (Quantitech, Qiagcn) and amplified material was detectedusing commercially available Taqman gene expression assays (AppliedBiosystems, Carlsbad, Calif.) with the data normalized to HPRT. Eachdata point represents 9 technical replicates from 3 independentbiological samples. Raw data of microarray studies are not yet availableat GEO worldwideweb.ncbi.nlm.nih.gov/geo).Animal Surgery. Rodent and monkey procedures were performed followingNIH guidelines, and were approved by the local Institutional Animal Careand Use Committee (IACUC), the Institutional Biosafety Committee (IBC)as well as the Embryonic Stem Cell Research Committee (ESCRO).Mice. NOD-SCID IL2Rgc null mice (20-35 g in weight; Jackson Laboratory,Bar Harbor, Me.) were anesthetized with Ketamine (90 mg/kg; Akorn,Decatur, Ill.) and Xylazine (4 mg/kg Fort Dodge, Iowa).6-hydroxydopamine (10 μg (ranging in concentration from 1-20 μg) 6-OHDA(Sigma-Aldrich) was injected stereotactically into the striatum at thefollowing coordinates (in millimeters): AP, 0.5 (from bregma; a skullsuture used as reference for stereotactic surgery); ML, −2.0; DV, −3.0(from dura a membrane covering the brain used for reference). Mice withsuccessful lesions (an average of >6 rotations/minutes) were selectedfor transplantation. A total of 150×10³ cells were injected in a volumeof 1.5 μl into the striatum at the following coordinates (in mm): AP,0.5; ML, −1.8; DV, 3.2. The mice were sacrificed 18 weeks posttransplantation.Rats. Adult female Sprague-Dawley (Taconic, Hudson, N.Y.) rats (180-230g) were anesthetized with Ketamine (90 mg/kg) and xylazine (4 mg/kg)during surgical procedures. Unilateral, medial forebrain bundle lesionsof the nigro-striatal pathway were established by stereotaxic injectionof 6-OHDA (3.6 mg/ml in 0.2% ascorbic acid and 0.9% saline, Sigma) attwo sites (Studer, et al. Nature Neurosci. 1:290-295 (1998), hereinincorporated by reference). Rats were selected for transplantation ifamphetamine-induced rotation exceeded 6 rotations/min by 6-8 weeks postinjection. 250×103 cells were transplanted into the striatum of eachanimal (Coordinates: AP+1.0 mm, ML −2.5 mm and V-4.7 mm; toothbar set at−2.5). Control rats received PBS instead. The surgical procedures weredescribed previously (Studer, et al. Nature Neurosci. 1:290-295 (1998),herein incorporated by reference). Daily intraperitoneal injections ofcyclosporine 15 mg/kg (Bedford Labs, Bedford, Ohio) were started 24hours prior to cell grafting and continued until sacrifice, 20 weeksfollowing cell grafting. Primates. Two adult (17-18 yr old; 10-12 kg;female) rhesus monkeys were rendered hemiparkinsonian via carotid MPTPadministration followed by weekly I.V. MPTP administration to create abilateral parkinsonian syndrome (Kordower, et al. Science 290:767-773(2000), herein incorporated by reference). Both animals displayedparkinsonian symptoms consistent with a moderately-severe lesion basedon behavioral analysis including crooked posture, dragging of leg andsymptoms of rigor (inflexibility of movement), neglect (motor awarenessto lateralized stimulus) and bradykinesia (slow movement intiation).These parameters can be assessed in monkeys using a modifiedParkinsonian clinical rating scale (CRS). On the day of transplantationsurgery, animals were tranquilized with ketamine (3.0 mg/kg, IM) anddexdomitor (0.02-0.04 mg/kg IM), intubated to maintain a stable airwayand anesthetized with isoflurane. They were then placed into astereotaxic frame for surgery. Both rhesus monkeys underwent a singlesurgery with three intracranial injections of human floor plate-derivedDA cultures based on stereotaxic coordinates (Paxinos, et al. The RhesusMonkey Brain in Stereotaxic Coordinates (Academic Press, 2000), hereinincorporated by reference). Bilateral injections of cells (10ul/injection; 125,000 cell/ul) were performed at three sites(1-posterior caudate, 2-pre-commissural putamen and overlying whitematter) for a total volume of 3 μl per hemisphere. An infusion pumpattached to a stereotaxic micromanipulator was utilized to deliver thecells at a rate of 1 μl/min though a 50 μl Hamilton syringe with 28 Gneedle. After the injections were completed, the needle was left inplace for an additional 2-5 minutes to allow the infusate to diffuse offthe needle tip before slowly retracting the syringe. Immediatelyfollowing surgery, the animals received analgesics (buprenex, 0.01 mg/kgIM, BID for 72 hours post surgery; meloxicam, 0.1 mg/kg SQ, SID for 72hours post surgery) as well as an antibiotic (cephazolin, 25 mg/kg IM,BID) until 72-hours post-surgery. The animals received cyclosporine A(Neoral, Sandimmune) orally (30 mg/kg tapered to 15 mg/kg) once dailybeginning 48-hrs prior to surgery until sacrifice, one month followingtransplantation.Behavioral Assays. Amphetamine-induced rotations (mice and rats) and thestepping test (rat) were carried out before transplantation and 4, 8,12, 18 weeks after transplantation. Rotation behavior in mice wasrecorded 10 min after i.p. injection of d-amphetamine (10 mg/kg, Sigma)and recorded for 30 minutes. Rotation behavior in rats was recorded 40min after i.p. injection of d-amphetamine (5 mg/kg) and automaticallyassessed by the TSE VideoMot2 system (Germany). The data were presentedas the average number of rotations per minute. The stepping test wasmodified from Blume, et al. Exp. Neurol. 219:208-211 (2009) and Crawley,et al. What's Wrong With My Mouse: Behavioral Phenotyping of Transgenicand Knockout Mice (Wiley-Liss, 2000), all of which are hereinincorporated by reference. In brief, each rat was placed on a flatsurface; its hind legs were lifted by gently holding up the tail toallow only the forepaws to touch the table. The experimenter pulled therat backwards 1 meter at a steady pace. Adjusting step numbers from bothcontralateral and ipsilateral forepaws were counted. Data was presentedas the percentage of contralateral/(contralateral+ipsilateral) adjustingsteps. The cylinder test was performed by placing each animal in a glasscylinder and counting the number of ipsilateral versus contralateral pawtouches (out of 20 touches) to the wall of the cylinder as describedpreviously (Tabar, et al. Nature Med. 14:379-381 (2008), hereinincorporated by reference). Tissue Processing for rodents and primatesare described below.Mice and Rats: Animals (mice and rats) received overdoses ofPentobarbital intraperitoneally (50 mg/kg) to induce deep anesthesia andwere perfused in 4% paraformaldehyde (PFA). Brains were extracted,post-fixed in 4% PFA then soaked in 30% sucrose solutions for 2-5 days.They were sectioned on a cryostat after embedding in O.C.T. compound(Sakura-Finetek, Torrance, Calif.).Primates: Animals were sacrificed under deep anesthesia with ketamine(10 mg/kg, Intramuscular (IM)) and pentobarbital (25 mg/kg, intravenous(IV)) via cardiac perfusion with heparinized 0.9% saline followed byfresh cold 4% PFA fixative (pH7.4). Immediately following primaryfixation, brains were removed from the skull and post-fixed in 4% PFA,free-floating, for 24-36 hrs. They were then rinsed and re-suspended in10% sucrose on a slow shaker at 4° C., and allowed to “sink”. Theprocess was then repeated in 20% sucrose followed by 30% sucrose. Wholebrains were cut coronally into 40 um serial sections on a frozen sledgemicrotome and stored free-floating in cryopreservative medium at −20°Celcius.Immunohistochemistry: Cells were fixed in 4% PFA and blocked with 1%bovine serum albumin (BSA) with 0.3% Triton. Brain tissue sections werewashed in cold PBS and processed similarly. Primary antibodies werediluted in 1-5% BSA or Normal Goat Serum and incubated according tomanufacturer recommendations. A comprehensive list of antibodies andsources is provided as Table 6. Appropriate Alexa488, Alexa555 andAlexa647-conjugated secondary antibodies (Molecular Probes, Carlsbad,Calif.) were used with 4′,6-diamidino-2-phenylindole (DAPI) nuclearcounterstain (Thermo Fisher, Rockford, Ill.). For some analysesbiotinylated secondary antibodies were used followed by visualizationvia DAB (3,3′-Diaminobenzidine) chromogen.HPLC Analysis. Reversed-phase HPLC with electrochemical detection formeasuring levels of dopamine, Homovanillic acid (HVA) and DOPAC(3,4-Dihydroxy-Phenylacetic Acid) was performed as described previously(Roy, et al. Nature Med. 12:1259-1268 (2006); Studer, et al. Brain Res.Bull. 41:143-150 (1996), all of which are herein incorporated byreference). Culture samples were collected in perchloric acid at day 65of differentiation. For some experiments DA was measured directly in themedium using the same detection system but following aluminum extractionof dopamine and its metabolites using a commercially available kit asdescribed previously (Studer, et al. Brain Res. Bull. 41:143-150 (1996),herein incorporated by reference).Electrophysiological recordings: Cultures were transferred to arecording chamber on an upright microscope equipped with a 40×water-immersion objective (Eclipse E600FN; Nikon); cultures wereperfused with saline containing in mM: 125 NaCl, 2.5 KCl, 25 NaHCO₃,1.25 NaH₂PO₄, 2 CaCl, 1 MgCl₂, and 25 glucose (34° C.; saturated with95% 0₂-5% CO₂; pH 7.4; 298 mOsm/L). The saline flow rate was 2-3 ml/minrunning through an in-line heater (SH-27B with TC-324B controller;Warner Instruments). Neurons were visualized by video microscopy with acooled-CCD digital camera (CoolSNAP ES², Photometrics, Roper Scientific,Tucson, Ariz.). Cells selected for electrophysiological recordings hadneuron-like shapes with fine branching neurites. Somatic whole-cellpatch-clamp recordings in current clamp configuration were performedwith a MultiClamp 700B amplifier (Molecular Devices). Signals werefiltered at 1-4 kHz and digitized at 5-20 kHz with a Digidata 1440A(Molecular Devices). Recording patch electrodes were fabricated fromfilamented horosilicate glass (Sutter Instruments) pulled on aFlaming-Brown puller (P-97, Sutter Instruments) and had resistances of4-6 MS) in the bath. Electrodes were filled with internal solutioncontaining in mM: 135 K-MeSO₄, 5 KCl, 5 HEPES, 0.25 EGTA, 10phosphocroeatine-di(tris), 2 ATP-Mg, and 0.5 GTP-Na (pH 7.3, osmolarityadjusted to 290-300 mOsm/L). The amplifier bridge circuit was adjustedto compensate for electrode resistance and monitored. Electrodecapacitance was compensated. When series resistance increased >20%during the recording, the data were discarded because increasedresistance suggested a partial technical failure during recordings.Cell Counts and Stereological Analyses. The percentages of markerpositive cells at the floor plate (day 11) FIG. 1, midbrain dopamineneuron precursor (day 25), FIG. 2 and mature DA neuron stages (day 50 orlater) FIGS. 3 and 11, were determined in samples derived from at least3 independent experiments each. Images for quantification were selectedin a uniform random manner and each image was scored first for thenumber of DAPI-positive nuclei, followed by counting the number of cellsexpressing the marker of interest. Data are presented as mean±SEM.Quantification of human cells (identified with anti-hNA) and TH+ neuronswithin grafts was performed on every tenth section where a graft wasidentifiable. Cell counts and graft volume was determined using theoptical fractionator's probe and the Cavalieri estimator using theStereo Investigator software (MBF bioscience, Vermont) as describedpreviously in Tabar, et al. Nat. Biotechnol. 23:601-606 (2005), hereinincorporated by reference. Data are presented as estimated total cellnumber and total graft volume+/−standard error of means (SEM).

The following formulations describe exemplary cell culture medium foruse in developing embodiments of the present inventions.

hESC medium for maintenance (1 liter): 800 mL DMEM/F12, 200 mL ofKnockout Serum Replacement, 5 mL of 200 mM L-Glutamine, 5 mL ofPen/Strep, 10 mL of 10 mM MEM minimum non-essential amino 15 acidssolution, 55 μM of 13-mercaptoethanol, and bFGF (final concentration is4 ng/mL).KSR medium for hESC differentiation (1 liter): 820 mL of Knock out DMEM,150 mL of Knock out Serum Replacement, 10 mL of 200 mM L-Glutamine, 10mL of Pen/Strep, 10 mL of 10 mM MEM, and 55 μM of 13-mercaptoethanol.N2 medium for hESC differentiation (1 liter): 985 ml dist. H₂0 withDMEM/F12 powder, 1.55 g of glucose (Sigma, cat. no. G7021), 2.00 g ofsodium bicarbonate (Sigma, cat. no. S5761), putrescine (100 uL aliquotof 1.61 g dissolved in 100 mL of distilled water; Sigma, cat. no.P5780), progesterone (20 uL aliquot of 0.032 g dissolved in 100 mL 100%ethanol; Sigma, cat. no. P8783), sodium selenite (60 uL aliquot of 0.5mM solution in distilled water; Bioshop Canada, cat. no. SEL888), and100 mg of transferrin (Celliance/Millipore, cat. no. 4452-01), and 25 mgof insulin (Sigma, cat. no. 16634) in 10 mL of 5 mM NaOH.Dulbecco's Modification of Eagles Medium (DMEM), with 10% FBS forpreparing PMEF ((primary mouse embryo fibroblast (PMEF)) feeder cells)(1 liter): 885 mL of DMEM, 100 mL of FBS, 10 mL of Pen/Strep, and 5 mLof L-Glutamin.Alpha Minimum Essential Medium (MEM) with 10% FBS for preparing MS-5feeder cell medium (1 liter): 890 mL of Alpha MEM, 100 mL of FBS, 10 mLof Pen/Strep Gelatin solution (500 ml): Dissolve 0.5 g of gelatin in 500ml of warm (50-60° C.) Milli-Q water. Cool to room temperature.

In general, the following is a brief summary for exemplary methods ofmonitoring the production of mature DA neurons for use in grafting (see,also, conditions shown in Table 7. Day 13 is a midbrain floor platestage, characterized by co-expression of FOXA2/LMX1A. In addition toexpression of FOXA2 and LMX1A, a loss of OCT4 expression and lack ofinduction of forebrain markers PAX6 and FOXG1 is found. Day 25 is themidbrain DA neuron precursor stage, characterized by the continuousexpression of FOXA2/LMX1A and expression of the neuronal (TUJ1) and DAmarkers (NURR1, TH). Proliferating, Ki67+ cells and the number of PAX6and FOXG1 forebrain neural precursors are monitored, where these markersare not desired. For unbiased and rapid quantification ofimmunofluorescence data, an Operetta (Perkin Elmer) High ContentMicroscope was used for measurements. qRT-PCR assays was also used foreach marker to confirm immunofluorescence data. In some embodiments,cell lines (cultures) passing these preliminary in vitro tests, are usedfor engraftment, see, Table 7. In some embodiments, mature DA neuronswere cryopreserved without serum at day 25 (ranging from day 20-day 25)in culture medium+7% DMSO (ranging from 3%-12%) until thawed for use inengraftment. In some embodiments, cell samples are stored in liquidnitrogen. In some embodiments, cells are stored in low temperaturefreezers. In other embodiments, cryoprotectants such as myoinositol,polyvinyl alcohol, serum replacement, caspasc inhibition compounds, arecontemplated for use in addition to DMSO. After thawing, cells aretested for viability, marker expression, etc., prior for use ingrafting. In some embodiments, thawed cells were tested for maintenanceof function in long-term in vitro and in vivo assays for monitoringfreezing and storage conditions.

B. Studies to identify additional factors for the generation offunctional DA neurons. Additional “drop out” and “add in” experimentsfor tissue culture components are contemplated for use in producingcells of the present inventions. For example, FGF8 was shown thatalthough its use resulted in cells of the present inventions, it was notrequired for production of these cells. These experiments will beextended to additional reagents, such as those listed in Table 8, asadditives to cell cultures along with the four “core” molecules thatresulted in DA neuronal cells of the present inventions, i.e. i)Alk4/5/7 (“TGFβ-)inhibitor (SB431542), ii) Alk2/3 (“BMP”)-inhibitor(LDN-193189), iii) Smoothened (“SHH”)-agonist (Purrmorphamine), and iv)GSK3β-inhibitor (CHIR99021).

As described herein, the use of SB431542 and LDN193189 showed efficientneural conversion of pluripotent stem cells while the addition ofPurmorphamine and CHIR99021 to these cells demonstrated midbrain floorplate induction. Other chemicals and recombinant proteins were or can beused to provide long-teim trophic support and/or acceleratedifferentiation. Some of these compounds will be used in further testsin order to define their roles in cell differentiation described herein.

For these experiments, performance of other compounds will be comparedto exemplary limits for DA neuron differentiation (Table 7) versus theuse of the 4 core-factors (Table 8).

These type of experiments are contemplated to define the minimal numberof factors needed for producing authentic DA neuronal cells of thepresent inventions.

C. Embodiments for establishing dose response curves for potentialproliferating contaminants (pluripotent hESCs, neural rosettes). Duringthe development of the present inventions, no teratomas or excessiveovergrowths were observed within the grafts up to at least 5 monthssurvival in vivo. In order to monitor safety for longer term studies inorder to reflect contemplated longitivity of grafts in humans, a cellnumber threshold is contemplated for determining a clinically relevantcontamination limit of problematic cell types, such as undifferentiatedhESCs which may develop into teratomas or primitive neuroectodermalprecursors capable of significant proliferation. Therefore, someembodiments are contemplated for further enrichment of dopaminergicneurons in cells for use in grafts, i.e. depleting contaminating celltypes prior to engraftment, see, Table 7 for exemplary limits.

The following ecribes exemplary standardized set of additional in assaysfor validating enhancement strategies. For hESCs, a pre-determined mixof undifferentiated (Oct4+/Nanog+) cells with hES derived DA neuronswill be used to monitor clinical symptoms suggestive of mass effectand/or animal death in animal experiments. A dose response of one hEScell per 10,000 hESC-derived DA cells, 1/5000, 1/1000 and 1/100 will beperformed. Cells will be injected intra-striatally and the animals willreceive immunosuppression. Rats will be monitored closely and will besacrificed upon manifestation of neurological symptoms or at a maximumof 6 months. The brains will be analyzed for graft volumes andcomposition as described herein. The cell ratios will adjusted until aclear in vivo threshold is established for the emergence of teratomas.For determining contaminating levels of primitive neuroectodermalprecursors, a similar strategy will be followed. The presence of earlyneural precursors have a significant potential for proliferation andbroad differentiation into central nervous system as well as peripheralnervous system (PNS) fates. Graft analysis will consist of IHC forrosette cells (PLZF expression), their CNS progeny (neural precursorsexpressing Nestin/Sox2 or forebrain precursors, expressing FoxGl) aswell as graft volumes and a proliferation index (% Ki67+ of totalsurviving cells).

IV. Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerativedisorder and is estimated to affect 4.1-4.6 million patients world-wide,a number predicted to more than double by 2030. It is the second mostcommon neurodegenerative disorder after Alzheimer's disease, affectingapproximately 1 million patients in the US with 60,000 new patientsdiagnosed each year. The disease has a major socioeconomic impactcausing significant morbidity and mortality, and the combined direct andindirect costs of PD, including health care cost and lost income, isestimated to be approximately $25 billion per year in the US alone.Currently there is no cure for Parkinson's disease (PD), an age-related,progressive and disabling disorder. PD is characterized pathologicallyby a selective loss of midbrain DA neurons in the substantia nigra. Afundamental characteristic of PD is therefore progressive, severe andirreversible loss of midbrain dopamine (DA) neurons resulting inultimately disabling motor dysfunction. While pharmacological,exercise-based, gene- and surgical therapies have been developed for PD,none of those approaches are yet able to restore proper DA neuronfunction. Long-term control of motor symptoms in patients often remainssuboptimal, and while recognizing the importance progressivenon-dopamine responsive motor and non-motor symptoms, the fundamentalissue of long term dopamine-responsive symptom control remains an areaof critical therapeutic need. Widespread pathology is recognized in PD,affecting both central and peripheral nervous systems, the cardinalfeatures of PD (bradykinesia, rigidity, and tremor partially) arefundamentally related to DA neuronal cell loss and aredopamine-responsive. Thus PD is contemplated for treatment usingneuronal cell replacement due to the rather selective loss of midbrainDA neurons that is responsible for most motor symptoms of the disease. ahealthy human brain harbors approximately one million DA neurons.Therefore, in one embodiment, DA neuron replacement is contemplated torequire a relatively small number of surviving cells as compared to mostother disorders in the CNS.

One challenge in developing a cell based therapy for PD was theidentification of an appropriate cell source for use in neuronalreplacement. This search has been going on for more than 30 years, withmany potential sources for DA neuron replacement were proposed (Kriks,Protocols for generating ES cell-derived dopamine neurons in Developmentand engineering of dopamine neurons (eds. Pasterkamp, R. J., Smidt, &Burbach) (Landes Biosciences, 2008; Fitzpatrick, et al., Antioxid.Redox. Signal. 11:2189-2208 (2009)). In the past, several of thosesources have progressed to early stage clinical trials includingcatecholaminergic cells from the adrenal medulla Madrazo, et al., N.Engl. J. Med. 316, 831-834 (1987), carotid body transplants (Arjona, etal., Neurosurgery 53: 321-328 (2003)), or encapsulated retinal pigmentepithelial cells (Spheramine trial Bakay, et al., Front Biosci. 9,592-602 (2004). However, those trials mostly failed to show clinicalefficacy and resulted in poor long-term survival and low DA release fromthe grafted cells. Another approach was the transplantation of fetalmidbrain DA neurons performed in over 300 patients worldwide (Brundin,et al., Prog. Brain Res. 184, 265-294 (2010); Lindvall, & Kokaia, J.Clin. Invest 120:29-40 (2010). Therapy using human fetal tissue in thesepatients demonstrated evidence of DA neuron survival and in vivo DArelease up to 10 or 20 years after transplantation in some patients.However, in many patients, fetal tissue transplantation fails to replaceDA neuronal function. Further, fetal tissue transplantation is plaguedby multiple challenges including low quantity and quality of donortissue, ethical and practical issues surrounding tissue acquisition, andthe poorly defined heterogeneous nature of transplanted cells, which aresome of the factors contributing to the variable clinical outcomes.Examples of fetal transplantation are described in; Mendez, et al.Nature Med. (2008)); Kordower, et al. N. Engl. J. Med. 332:1118-1124(1995); Piccini, et al. Nature Neuroscience 2:1137-1140 (1999). However,the clinical results were mixed with some positive data in early,open-label studies (Lindvall, et al. Science 247:574-577 (1990); Widner,et al. N. Engl. J. Med. 327:1556-1563 (1992); Brundin, et al. Brain123:1380-1390 (2000); Freed, et al. N. Engl. J. Med. 327:1549-1555(1992); Freeman, et al. Bilateral fetal nigral transplantation into thepostcommissural putamen in Parkinson's disease. Ann Neurol 38:379-388(1995). However modest results were found in two larger, NIH-sponsored,placebo-controlled clinical trials in the US (Freed, et al. N. Engl. J.Med. 344, 710-719 (2001); Olanow, et al. Ann. Neurol. 54:403-414(2003)). There are many hypotheses as to the limited efficacy observedin the human fetal grafting trials including that fetal grafting may notprovide a sufficient number of cells at the correct developmental stagefor an optimal therapeutic benefit. Furthermore, fetal tissue is quitepoorly defined by cell type and variable with regard to the stage andquality of each tissue sample Bjorklund, et al. Lancet Neurol. 2,437-445 (2003). Another contributing factor may be a low-levelinflammatory host response to the graft (Bjorklund, et al. LancetNeurol. 2, 437-445 (2003)).

In contrast, a stem cell-derived cell source or other type of consistentcell type for use in providing cells for transplantation is contemplatedto overcome many of the challenges associated with fetal tissue graftingand could offer an unlimited source of DA neurons at the optimum stagefor transplantation. After nearly 20 years of attempts using variouspotential stem cell sources, the inventors' succeeded in obtainingauthentic human midbrain DA neurons from pluripotent stem cells capableof reversing neurological defects in murine animals and primates. Thisnovel differentiation strategy was highly efficient and led to robust invivo engraftment of the cells, induction of functional recovery in PDmodels of disease, and lack of adverse events such as inappropriate cellproliferation as supported by preclinical data. FDA approval for a humanES cell based strategy for treating PD is contemplated because the FDAapproved testing other human ES cell derivatives in spinal cord injury(Strauss, Nat. Biotechnol. 28:989-990 (2010) and macular degeneration(Schwartz, et al. Lancet 379:713-720 (2012)).

Additional embodiments including “enhancement” strategies to controlcell purity promote axonal fiber outgrowth and include novelsafety/regulatory features in grafting strategies are contemplated. Forexample, in some embodiments, a method of cell purification demonstratedstarting from a simple surface marker screen against a cell type ofinterest, (such as CD142) towards a meaningful enrichment strategy for aspecific neuron type. In some embodiments, this method is contemplatedfor use in providing cells for use in humans. Further, in otherembodiments, engineered expression of PSA-NCAM is contemplated forenhancing axonal outgrowth for use in neural repair in vivo. Suchapplications include promotion of long-distance axonal growth fortreating motoneuron disease, Huntington's disease or other disordersprimarily affecting projection neurons. Additional embodiments arecontemplated for a GMP qualified pluripotent cell source, and the like.Because the engraftment methods described herein, require a small numberof DA neurons and are based upon relatively simple, cost-effective smallmolecule methods developed for DA neuron induction, it is contemplatedthat DA neuron replacement therapy would be at a reasonable cost on aper patient basis.

One potential biological limitation of the transplantation approach inPD is the fact that neuronal degeneration in PD proceeds to affect manycell types other than midbrain dopamine neurons, particularly at laterstages of the disease. In fact in a long term study, non-DA responsivesymptoms predominate in late PD, leading to dysphagia, falls, dementiaand other significant morbidities. However, some non-motor symptoms arecontemplated to benefit from restoring dopaminergic function.Furthermore, it is contemplated that the use of hESC derived DA neuronsat early stages of the disease would prevent some of the secondary PDsymptoms, including the degeneration of the dopamine receptivepopulations of the striatum. However, even in the absence of impactingthe non-DA responsive symptoms of the disease, the long-term functionaldopaminergic restoration of the striatum would be a major achievementfor treating this currently incurable disorder. In the case ofParkinson's disease there are several alternative therapies availableincluding drug-based strategies and surgical approaches such as deepbrain stimulation. In some embodiments, efficacy of recovery iscontemplated to be comparable to or beyond the levels achieved withalternative therapies. In other embodiments, use of this mature DAneuron cell engraftment therapy is contemplated to be particularlybeneficial for a particular subset of patients. In other embodiments,use of this mature DA neuron cell engraftment therapy is contemplatedfor use in addition to existing drug and surgical type approaches. Onemajor benefit of using mature DA neuron cell engraftment therapy of thepresent inventions is the unique neurorestorative nature postengraftment, i.e. long term recovery of neuronal function that iscontemplated for use in patients for progressive removal of drugtherapy. Cell transplantation is contemplated to affect a differentspectrum of DA-related symptoms than those responding to drugs or othertherapy. Thus in one embodiment, mature DA neuron transplantation iscontemplated for use with DBS. In another embodiment, mature DA neuronis contemplated for use with therapy.

A) Parkinson's Disease and Current Therapies. Great progress was made inthe identification of rare genetic changes contributing to familialforms of PD. However, for the majority of PD cases the contribution ofany potential genetic predisposition remains unclear. Traditionaltherapeutic strategies in PD are limited by the fact that at the onsetof clinical symptoms 30-70% of all DA neurons in the substantia nigrahave irreversibly degenerated. One therapeutic option is thepharmacological replacement of DA neuron deficiency using the DAprecursor L-Dopa. However, despite the dramatic initial response of somePD patients to L-Dopa therapy, long-term clinical outcome remains poorand severe side effects of L-Dopa therapy, including motor fluctuationsand dyskinesias, occur frequently in late-stage disease. Pulsatiledelivery of L-dopa has a major role in development of these later stagemotor complications therefore a “smoother” more physiologic delivery ofdopamine, i.e. such as from engrafted cells of the present inventions,would therefore be highly desirable. In addition to pharmacologicalstrategies, there are several surgical treatment options. These includethe ablation or functional inactivation of cells within the basalganglia via pallidotomy or deep brain stimulation by targeting thesubthalamic nucleus or globus pallidus pars interna. While thesesurgical options are alternatives for some patients, they providesymptomatic relief from the disease but do not restore normal DAfunction. Furthermore surgical and non-surgical side effects have beenreported, including hardware malfunction, infections, stroke, hemorrhageand the like. Other treatment options include the delivery of growthfactors such as GDNF or Neurturin using direct intra-parenchymalinfusion or viral expression by gene therapy. While initial open labelstudies in PD showed promising results for GDNF subsequent controlledtrials in a larger set of patients failed to confirm any benefit andraised potential safety concerns due to the production of anti-GDNFantibodies in a subset of patients. AAV-based delivery of Neurturin, amolecule related to GDNF, also failed to show any significant clinicalbenefit in a large placebo-controlled, multicenter trial. Early datafrom a Phase 2 trial of AAV-borne glutamic acid decarboxylase (GAD)injections into the subthalamic nucleus were recently reported (LancetNeurology, April 2011) however, the clinical benefits were modest atbest in this study. While efforts on neurotrophic factor-based- oralternative neuroprotective strategies might bring temporary relief topatients, none of them can bring back/replace DA neurons already lostdue to the disease, the main goal a cell replacement therapy.

B) Cell therapy in PD. Clinical symptoms become apparent in PD after70-80% of striatal dopamine and about 50% of nigral dopamine neurons arelost. However, midbrain dopamine neurons developed by 8.5 weeks postconception with little evidence of dopamine neuron replacementthroughout the remainder of life. Therefore, dopamine neurons by time ofdisease onset are many decades old without a natural mechanism toreplace these cells, thus cell transplantation may be needed to replacethose cells in the brains of PD patients. DA neuron replacement in PDwas done in the 1980s based on the use of adrenal medulla derivedchromaffin cells. Those hatinone producing cells were shown to switchneurotransmitter phenotype from adrenalin to DA when placed ectopicallyinto the CNS. While several hundred PD patients were grafted worldwideusing this approach, it became clear over time that grafted cellssurvive very poorly with a transient effect at best. Therefore, thisapproach was quickly abandoned for clinical use. In contrast, the use offetal midbrain tissue grafting was based on more extensive preclinicalstudies in rodent models that demonstrated robust long-term engraftmentand functional improvement across a panel of DA related behavioralassays. Encouraged by those preclinical data, fetal grafting proceededat multiple centers in the late 1980s and early 1990s. Those studiesshowed clear evidence that functional long-term engraftment withincreased DA release in the grafted area as measured by Fluorodopa PETand subsequent histological studies in some patients that died due tounrelated causes. However, the use of fetal graft raised two potentialproblems with cell-based therapeutic approaches. First, an unexpectedproblem of fetal grafts was the induction of graft-induced dyskinesias(GID) in about 15% of patients. While the mechanism of GID remainscontroversial, recent evidence indicated that serotonergic neurons werecapable of inappropriate storage and release of DA. Another potentialmechanism suggested to explain GID was the uneven distribution of DAneurons, i.e. causing hot spots of DA release. In contrast, during thedevelopment of the present inventions, methods for detecting andreducing Serotonergic neurons were discovered which would reduce theincidence of GID. Further, injection of mature DA neurons would providean even distribution of mature neurons including extending dopaminergicfiber terminals within the host striatum. Another problem with fetalgrafting treatment was (and is) limited availability of fetal midbraintissue at the appropriate developmental stage. An alternative strategywas tried clinically to address the issue of limited supply by usingfetal pig derived DA neurons. However, DA neuron survival in thosexenografts was poor and the overall approach was abandoned. A recenttrial using retinal pigmented epithelium also failed to show anybenefits. In contrast, the use of human ES cells as sources oftransplant cells is contemplated to provide an unlimited source of cellsfor making dopamine neurons for use in transplantation.

V. Compounds and Culture Methods were Discovered for DirectedDifferentiation of FOXA2+ and LMX1A+ Positive Neuronal Precursor Cellsinto Midbrain DA (mDA) Neurons of the Present Inventions.

The inventors' discovered during the development of the presentinventions that timing of CHIR99021 exposure determines induction ofFOXA2/LMX1A midbrain floor plate precursors. Therefore the inventorstested for immunocytochemical analysis of FOXA2/LMX1A at day 11 ofdifferentiation following LSB/S/F8 (i.e. treating cells with LSB, S,i.e. SHH, and FGF8 (F8) treatment alone or in combination with CHIRstarting at various timepoints: d(day)0-d11, dl-d11, d3-d11, d5-d11,d7-dl 1 compared to duplicate cultures of cells with no CHIR treatment.Then quantification of the percentage of FOXA2+, LMX1A+ and doublelabeled cells were determined at day 11 of differentiation followingdifferential onset of CHIR exposure as described in theimmunocytochemical analysis.

A. CHIR99021 (C) Is A Factor For Inducing FOXA2+/LMX1A+ Cells By Day 11From LSB Cultured Cells Contacted With An activator of Hedgehog andPurmorphamine. The following example describes using exemplary methodsfor testing the efficacy of each compound for inducing directed neuronaldifferentiation of mDA neurons.

This example describes the discovery of small molecules and contacttiming for providing directed differentiation of FOXA2+LMXIA+DA neuronsof the present inventions. The following is a brief summary of some ofthe experimental discoveries described herein: Treatment of Dual-SMADinhibited cells with SHH agonists (purmorphamine+SHH) and FGF8 (S/F8) inthe absence of CHIR99021 showed significantly lower expression of FOXA2by day 11 and complete lack of LMX1A expression (FIG. 1a,b ). Theanterior marker OTX2 was robustly induced in LSB and LSB/S/F8/CHIRtreated cultures, but not under LSB/S/F8 conditions (FIG. 1a,c ).

A cell population containing pluripotent cells was chosen by theinventors for a starting population and plated at Day 0. Cell are grownto near confluency prior to differentiation (between 60-100%confluence). These cells were contacted with Dual SMAD inhibitors (i.e.exposure to LDN-193189+SB431542=“LSB”) on Day 0. The inventors followeda cell population with regular feedings containing fresh LSB until Day11 and discovered that some remaining cells were LMX1A+ but did notexpress FOXA2 (FIG. 1a,b ). The inventors plated duplicate starting cellpopulations then tested for cell types (i.e. gene/protein expressionpatterns) after contacting with mixtures containing any of the followingSHH agonists (purmorphamine+SHH) and FGF8 (S/F8) contacting the cellswith different exposure regimens, i.e. contacting cells at Day 0, or Day1, or Day 2, etc. for specific amounts of time, i.e. 24 hours, 48 hours,etc. Three primary exemplary culture conditions tested were 1) cellscontacted with LDN/SB (LSB) on Day 0 then contacted with fresh LSB untilDay 5, on Day 5 cells were contacted with fresh LDN without SB until Day11, 2) cells contacted with LDN/SB (LSB) on Day 0 then contacted withfresh LSB until Day 5, on Day 5 cells were contacted with fresh LDNwithout SB until Day 11 while during this time cells were additionallycontacted with fresh purmorphamine, SHH and FGF8 until Day 7 and 3)cells contacted with LDN/SB (LSB) on Day 0 then contacted with fresh LSBuntil Day 5, on Day 5 cells were contacted with fresh LDN without SBuntil Day 11 while during this time cells were additionally contactedwith fresh purmorphamine, SHH and FGF8 until Day 7 while additionallycontacted with fresh CHIR starting on Day 3 of culture until Day 11 withseveral variations of these primary conditions in order to determineoptimal yield of cell types.

B. In Vitro Characterization Of FOXA2+/LMX1A+ Cells Derived From TheMidbrain Region Of The Floor Plate In Comparison To DA Precursor CellsGenerated With Other Techniques. Systematic comparisons of the threeculture conditions (FIG. 1d ) were performed using global temporal geneexpression profiling. Hierarchical clustering of differentiallyexpressed genes segregated the three treatment conditions by day 11 ofdifferentiation (FIG. 8a ). FOXA1, FOXA2 and several other SHHdownstream targets including PTCH1 were amongst the most differentiallyregulated transcripts in LSB/S/F8/CHIR versus LSB treatment sets (FIG.1e ). Expression of LMXIA, NGN2, and DDC indicated establishment ofmidbrain DA neuron precursor fate already by day 11 (FIG. 1e,f ). Incontrast, LSB cultures were enriched for dorsal forebrain precursormarkers such as HES5, PAX6, LHX2, and EMX2. Direct comparison ofLSB/S/F8/CHIR versus LSB/S/F8 treatment (FIG. 1f ) confirmed selectiveenrichment for midbrain DA precursor markers in LSB/S/F8/CHIR group andsuggested hypothalamic precursor identity in LSB/S/F8 treated culturesbased on the differential expression of RAX1, SIX3, and SIX6 (see alsoPOMC, OTP expression in FIG. 2d below). An exemplary list ofdifferentially expressed transcripts are shown, i.e. Tables 1, 2 andgene ontology analysis for Day 11, FIG. 8b (DAVID;http://david.abcc.ncifcrf.gov) confirmed enrichment for canonical WNTsignaling upon CHIR treatment. Raw data are not yet available at GEOworldwideweb.nebi.nlm.nih.gov/geo/accession#: [TBD]). Comparativetemporal analysis of gene expression for midbrain DA precursor markers(FIG. 1g ) versus markers of anterior and ventral non-DA neuron fates(FIG. 1h ) partitioned the three induction conditions into: i) LSB:dorsal forebrain; ii) LSB/S/F8: ventral/hypothalamic and iii)LSB/S/F8/CHIR: midbrain DA precursor identity.

VI. Further Differentiation Of FOXA2+/LMX1A+ Day 11 Cells Into MidbrainDA Neurons By Day 25 And Maintained Up To Day 65.

For further differentiation, precursor FOXA2+/LMX1A+ cells weremaintained in a medium promoting neuronal maturation (BAGCT, see ExampleI). For comparison two other techniques were used to generate DAneuronal precursor cells. The following types of comparisons were madebetween the populations of differentiated cells resulting from previousmethods and methods of the present inventions: A) Immunocytochemicalanalysis at day 50 of differentiation for TH in combination with LMX1A,FOXA2 and NURR1, B) Quantification of TH+, FOXA2+, LMX1+, and NURR1+cells out of total cells comparing rosette-derived versus floorplate-derived (LSB/S/F8/CHIR) cultures. C) Quantification of thepercentages of serotonin+(5-HT), and GABA+ neuronal subtypes (non-DAneuron contaminants) at day 50 in floor plate and rosette-derived DAneuron cultures. And D) HPLC analysis for measuring dopamine andmetabolites: Comparison of the DA, DOPAC and HVA levels between floorplate versus rosette-derived cultures.

By day 25, three precursor cell populations yielded Tuj1+ neurons (FIG.2a ) and cells expressing TH, the rate-limiting enzyme in the synthesisof DA. However, LSB/S/F8/CHIR treatment yielded TH+ cells thatco-expressed LMX1A and FOXA2 and displayed strong induction of thenuclear receptor NURR1 (NR4A2) (FIG. 2a,b ). Comparing gene expressionin day 13 versus day 25 cultures confirmed robust induction of otherpostmitotic DA neuron markers (FIG. 2c ). Characterizing DA neuronidentity at day 25 in comparison to LSB and LSB/S/F8 treated culturesconfirmed enrichment for known midbrain DA neuron transcripts andidentified multiple novel candidate markers (FIG. 2d , Tables 3-5, FIG.8b ). For example, the transcript most highly enriched in LSB/S/F8/CHIR(midbrain DA group) was TTF3, a gene not previously associated withmidbrain DA neuron development, but highly expressed in the humansubstantia nigra (FIG. 8c ; Allen Brain Atlas:http://human.brain-map.org).

Similar data were obtained for EBF-1, EBF-3 (FIG. 8c ) as well as TTR, aknown transcriptional target of FOXA2 in the liver. The data obtainedduring the development of the present inventions indicated enrichment ofseveral PITX genes in midbrain DA precursor cells. PITX3, a classicmarker of midbrain DA neurons, was also robustly expressed at day 25 ofdifferentiation (FIG. 2e ). Finally, both midbrain floor plate and DAneuron induction could be readily reproduced in independent hESC andhiPSC lines (FIG. 9). The data demonstrated herein showed that theLSB/S/F8/CHIR protocol as opposed to other tested protocols yields cellsexpressing a marker profile matching midbrain DA neuron fate.

In vitro and in vivo properties of floor plate-derived DA neurons werecompared to DA-like neurons obtained via a neural rosette intermediate(FIGS. 10 and 16). Patterning of neural rosettes represents thecurrently most widely used strategy for deriving DA neurons from hPSCs.Both floor plate- and rosette-based protocols were efficient atgenerating TH+ neurons capable of long-term in vitro survival (day 50 ofdifferentiation; FIG. 3-1 a). However, the percentage of TH+ cells wassignificantly higher in floor plate-derived cultures (FIG. 3-1 b). WhileTH+ cells in both protocols displayed co-expression of NURR1, floorplate-derived DA neurons co-expressed FOXA2 and LMX1A (FIG. 3-1 a,b and3-2).

Few GABA and serotonin (5-HT)-positive neurons were observed (FIG. 3-1c). DA, and its metabolites DOPAC and HVA, were present in culturesgenerated with either protocol, but DA levels were approximately 8 timeshigher in floor plate cultures (FIG. 3-1 d,e). Midbrain DA neuronsexhibited extensive fiber outgrowth and robust expression of matureneuronal markers including synapsin, dopamine transporter (DAT), andG-protein coupled, inwardly rectifying potassium channel (Kir3.2, alsocalled GIRK2, expressed in substantia nigra pars compacta (SNpc) DAneurons) (FIG. 3-1 f, FIG. 11). SNpc DA neurons in vivo exhibit anelectrophysiological phenotype that differentiates them from most otherneurons in the brain. In particular, they spike spontaneously at a slow(1-3 Hz) rate. Moreover, this slow spiking is accompanied by a slow,sub-threshold oscillatory potential. After 2-3 weeks in vitro, thesesame physiological features are displayed by SNpc DA neurons culturedfrom early postnatal mice. The DA neurons differentiated from hESCsconsistently (4/4 tests) displayed this distinctive physiologicalphenotype (FIG. 3-1 g-i).

Maintenance of mDA neurons in vitro at d65 showed TH positive neuronsare still expressing FoxA2 and extend long fibers typical for mDAneurons. FIG. 3-lj. DA release measurement by HPLC showed d65 old TH+neurons are functional in vitro FIG. 3-1 k.

In summary, neurogenic conversion of midbrain floor plate precursors andthe development of an optimized floor plate midbrain DA neurondifferentiation protocol is described herein. Floor plate derived DAneurons were obtained from human ES cells following small molecule basedactivation of SHH and canonical WNT signaling during earlydifferentiation stages (FIG. 3-2). These hES cells progressed from aFOXA2/LMX1A double positive midbrain floor plate stage, to Tuj1+immature neurons with co-expression of FOXA2/LMX1A then to mature DAneurons (FIG. 3-2 a,b) with robust DA release and electrophysiologicalproperties characteristic of substantia nigra pars compacta (SNpc;A9-type) midbrain DA neurons, including autonomous pacemaking activity(FIG. 3-2 c). Surprisingly, this was a highly efficient process withmore than half of cells in the culture dish adopting mature midbrainmarker profile (see FIG. 3-2 b).

VII. Characterization of Floor Plate-Derived Midbrain Dopamine NeuronsIn Vivo as Engrafted Neurons.

The following example describes using exemplary methods of the presentinventions for use in therapeutic cell replacement. One major challengein the field is the ability to generate hPSC-derived midbrain DA neuronsthat functionally engraft in vivo without the risk of neural overgrowthor inappropriate differentiation into non-midbrain neurons or developteratomas. Based on fetal tissue transplantation studies, the inventors'contemplated that the time of cell cycle exit, marked by expression ofNURR1, may be a suitable stage for grafting (approximately day 25 ofdifferentiation, FIG. 2). Initial studies using day 25 cells innon-lesioned adult mice showed robust survival of hPSC-derivedFOXA2+/TH+ neurons at 6 weeks after transplantation (FIG. 12). Survivalof FOXA2+/TH+ cells long-term in Parkinsonian hosts without resulting inneural overgrowth was tested. To this end, 6-hydroxy-dopamine (6-OHDA)lesions (Tabar, et al. Nature Med. 14:379-381 (2008), hereinincorporated by reference) were made in NOD-SCID IL2Rgc null mice, astrain that efficiently supports xenograft survival with particularsensitivity for exposing rare tumorigenic cells (Quintana, et al.Efficient tumour formation by single human melanoma cells. Nature456:593-598 (2008), herein incorporated by reference). Both floor plate-and rosette-derived DA neuron cultures were grafted (150×10³cells/animal) without prior purification in order to reveal potentialcontaminating cells with high proliferative potential. Four and a halfmonths after transplantation floor plate-derived DA neuron grafts showeda well-defined graft core composed of TH+ cells co-expressing FOXA2 andthe human specific marker hNCAM (FIG. 4a-c ). Functional analysis showeda complete rescue of amphetamine-induced rotation behavior. In contrast,rosette-derived neuronal grafts showed few TH+ neurons, did not producea significant reduction in rotation behavior (FIG. 4d ) and displayedmassive neural overgrowth (graft volume >20 mm³; FIG. 13). Extensiveovergrowth of rosette-derived neuronal cells used in grafting asreported herein was comparable to previous work with rosette-derived DAgrafts from the inventors' group (Kim, et al. miR-371-3 ExpressionPredicts Neural Differentiation Propensity in Human Pluripotent StemCells. Cell Stem Cell 8:695-706 (2011), herein incorporated byreference) and others (Hargus, et al. Proceedings of the NationalAcademy of Sciences of the United States of America 107:15921-15926(2010), herein incorporated by reference). The overgrowth was likely dueto the longer survival periods (4.5 months versus 6 weeks), lack of FACSpurification prior to transplantation and choice of NOD-SCID IL2Rgc nullhost. The number of proliferating Ki67+ cells was minimal in floorplate-derived grafts (<1% of total cells), while rosette-derived graftsretained pockets of proliferating neural precursors. Neural overgrowthis thought to be caused by primitive anterior neuroectodermal cellswithin the graft (Elkabetz, et al. Genes Dev. 22:152-165 (2008); Aubry,et al. Proc. Natl. Acad. Sci. USA 105:16707-16712 (2008), hereinincorporated by reference). This hypothesis was supported by theexpression of the forebrain marker FOXG1 in rosette-but not floorplate-derived grafts. A small percentage of astroglial cells werepresent in both floor plate- and rosette-derived grafts, though mostGFAP+ cells were negative for human markers indicating host origin (FIG.13).

Results in NOD-SCID IL2Rgc null mice described herein demonstratedrobust long-term survival of FOXA2+/TH+ neurons, complete reversal ofamphetamine-induced rotation behavior and lack of any signs of neuralovergrowth. However, some of these outcomes could be attributable to thespecific use of NOD-SCID IL2Rgc null mice. To test this hypothesis,floor plate-derived DA neuron cultures (250×10³ cells) were transplantedin adult 6-OHDA lesioned rats immunosuppressed pharmacologically usingcyclosporine A. Five months after transplantation graft survival wasrobust (FIG. 4e-h ) with an average of more than 15,000 TH+ cellsco-expressing FOXA2 (FIG. 4g ), and human nuclear antigen (hNA) (FIG. 4e); TH+/hNCAM+ fibers emanated from the graft core into the surroundinghost striatum (FIG. 4f ). In addition to FOXA2, TH+ cells expressedmidbrain DA neuron markers PITX3 and NURR1 (FIG. 4h-j ). Behavioralanalyses showed complete rescue of amphetamine-induced rotationalasymmetry, in contrast to sham-grafted animals that did not showimprovements (FIG. 4k ). Grafted animals also showed improvements in thestepping test (FIG. 4l ) measuring forelimb akinesia and in the cylindertest (FIG. 4m ), assays that do not depend on pharmacologicalstimulation of the DA system. The late onset of recovery (approximately3-4 months after transplantation) is expected for human DA neurons anddepends on the rate of in vivo maturation such as the levels of DATexpression (FIG. 4n ). The presence of TH+ cells expressing Kir3.2channels (GIRK2) or calbindin indicate that both SNpe (A9) and ventraltegmental area (A10) DA neurons are present in the graft (FIG. 4o,p ).

As in mice (FIG. 13), serotonergic and GABAergic cells were rare (<1% oftotal cells) in rat cells, as were the mostly host-derived GFAP+ glialcells (7% of total cells; (FIG. 14). While few serotonin+ neurons weredetected in the graft, hNCAM-negative cells were observed that werelikely host-derived serotonergic fibers (FIG. 14).

Engraftment of floor-plate derived DA neurons in mice, rats, and monkeysdemonstrated the surprising recovery of neuronal function in rodent andprimate species. Short-term (6 weeks) survival assays were extended forsurprisingly long-term survival for up to 5 months after transplantationinto the mouse striatum of 6OHDA lesioned and immunocompromised hostmice. In fact, a direct comparison of a traditional, rosette-basedmethod of making DA neurons (Perrier, et al. Proc Natl Acad Sci USA 101,12543-8 (2004)) compared to the novel floor plate based DA neurondifferentiation protocol described herein, showed that floor platederived DA neurons were capable of long-term DA neuron engraftment whilerosette-based neurons were not (FIG. 4a-c ).

In particular, a robust induction of behavioral recovery inamphetamine-induced rotations in 6-OH lesioned mice transplanted withfloor-plate (fp)-derived (blue line) neurons and rosette-derived (redline) DA neurons (FIG. 4d ) showed that fp derived neurons had higherrecovery rates. Mice grafted with floor-plate derived DA neurons showedalmost complete recovery in amphetamine scores. Animals grafted withrosette-derived DA neurons showed less behavioral improvement and someover time reversed to initial high rotation numbers.

Robust graft function was also found in the 6OHDA lesioned rat model.The rat, unlike the PD mouse, allows for more complex behavioral assaysand addresses DA neuron survival in a xenografting setting followingpharmacological immunosuppression (a therapy more closely mimickinghuman grafting protocols). Excellent graft survival, evidence of DAfiber outgrowth and maintenance of midbrain specific transcriptionfactor expression confirmed long-term survival of floor-plated derivedneurons expressing authentic midbrain DA neuron markers (FIG. 4e-j ). Abattery of functional assays showed significant improvement in both druginduced (amphetamine-induced rotations) and in spontaneous behavioraltests (cylinder and stepping test).

The results demonstrated herein showed excellent graft survival andbehavioral outcome in two independent murine models. However, the numberof DA neurons required in a mouse or rat brain represents a smallfraction of the larger number of cells needed for engrafting in primatesand humans. To test the scalability of this protocol, performed pilotgrafting studies were done in two adult MPTP lesioned rhesus monkeys.

Additionally, it was not initially known whether the methods and cellsdescribed herein would also restore neuronal function in a primate,information which might be used in support of enablement for use ofthese methods and cells in humans. Thus, an initial set of studies wasperformed in at least 2 monkeys to test short-term (4-6 weeks) in vivosurvival and maintenance of midbrain DA neuron phenotype in the primatebrain. Those studies described herein, showed robust survival ofTH/FOXA2 positive midbrain DA neurons and evidence of re-innervation ofthe host striatum (FIG. 4q-t ). Grafting larger numbers of cells,similar to those estimated in the number range required for future humangrafting studies, resulted in robust midbrain DA neuron survival. Inaddition to these short-term data, a set of longer-term studies inrhesus monkeys was done evaluating 3 month-survival of cells and anoptimized immunosuppression regimen of CellCept, Prograf, and Prednisonedaily (used as triple therapy in combination). Surprisingly, robust 3month survival of human ES derived midbrain DA neurons in the primatebrain was discovered along with a greatly reduced inflammatory hostreaction to the grafts compared to the strong host microglia responseobserved in the initial grafting study (see, FIG. 15).

Specifically, methods relating to the monkey studies included, batchesof 50×10⁶ transplantable DA neuron precursors were obtained by day 25 ofdifferentiation using the floor plate-based protocol. Classic dose was 3mg MPTP-HCL injected into the carotid artery (range 0.5-5 mg). This wasfollowed by systemic injection of MPTP 0.2 mg/kg IV of MPTP. Cells wereinjected at three locations (posterior caudate and pre-commissuralputamen) on each side of the brain (6 tracts in total, 1.25×10⁶cells/tract), and the animals were immunosuppressed with cyclosporine-A.One side of the brain was injected with DA precursors from a GFPexpressing subclone of H9, while the other side was engrafted with cellsderived from unmarked H9 cells. Results showing engraftment of neuronsin rhesus monkeys with continued FOX2A expression and TH production areshown in FIG. 4q-t . One month after transplantation, robust survival ofmidbrain DA neurons was observed based on expression of GFP (FIG. 15)and the human specific cytoplasmic marker (SC-121) (FIG. 4q ). Eachgraft core was surrounded by a halo of TH+ fibers extending up to 3 mminto the host (FIG. 4r ). The graft cores were composed of TH+ neuronsco-expressing SC-121 (FIG. 4s ) and FOXA2 (FIG. 4t ). SC-121 and GFPnegative areas within the graft contained Iba1+host microglia (FIG. 15)indicating incomplete immunosuppression.

In summary, engraftment of novel DA neuronal cell population inprimates, i.e. adult MPTP (3 mg of of MPTP-HCL(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ranging in concentrationfrom 0.5-5 mg MPTP-HCl) lesioned rhesus monkeys containing a severe >95%loss of endogenous midbrain DA neurons. MPTP exposure caused observablechanges and symptoms similar to Parkinson's disease in humans.

In summary, a novel floor plate-based hPSC differentiation protocol wasdiscovered that faithfully recapitulates midbrain DA neuron development.Access to cells with the cardinal features of midbrain DA neurons willenable a broad range of biomedical applications such as basicdevelopmental studies, high-throughput drug discovery and PD-iPSC baseddisease modeling. Importantly, this study finally established a means ofobtaining a scalable source of FOXA2+/TH+ neurons for neuraltransplantation—a major step on the road towards considering a cellbased therapy for PD.

Furthermore, derivation of authentic midbrain DA neurons from hESCsshowed excellent in vivo performance (see FIG. 4), and DA neuron yield,at the time of grafting (approximately 40% mature neurons in thepopulation at Day 25-30 of differentiation, that exceeded thepercentages obtained following dissection of human fetal ventralmidbrain tissue (typically approximately 10%) (Sauer, et al., Reston.Neurol. Neurosci. 2:123-135 (1991)).

The following are brief descriptions of materials and methods for use inevaluating two main parameters after engraftment: duration of survivaland extent of behavioral assessments. For short term studies e.g. aimingat confirming cell survival or phenotypic composition, behavioralassessments are contemplated and the animals will be tested about 4-8weeks post grafting for survival. Long-term studies will includebehavioral assessment post grafting and animal survival for at least 5months following grafting. When analyzing enhancement strategies, suchas PSA-NCAM modifications, behavioral assessment are contemplated toinclude more complex parameters such as the staircase test for skilledforelimb use.

Exemplary protocols for assessment of in vivo performance has at leastfour main components and includes the following: i—Lesion Induction,ii—Core Behavioral Analysis, iii Grafting, and iv—Tissue Analysis.Although these procedures were used on rats, in some embodiments theseprocedures may be use on other species.

i-Lesion Induction. Unilateral injections of 6-hydroxydopamine (6-OHDA)in the median forebrain bundle (MFB) is one standard approach forinduction of Parkinson-like symptoms in rats. 6-OHDA is a neurotoxinthat gets retrogradely transported via the nigrostriatal pathway in theMFB back to the substantia nigra whereby it results in neuronal celldeath via impairment of mitochondrial respiratory enzymes. Targetedneurons include the A9 dopaminergic neurons within the substantia nigracompacta (SNC) as well as the A10 neurons in the ventral tegmental area.This lesion model is widely studied and accepted as an excellentpre-clinical model for the study of the neurochemical and behavioralconsequences of advanced PD as it results in extensive unilateraldepletion of dopamine within the caudate-putamen complex (CPU). Thebehavioral consequences are also well described and include spontaneousand drug-induced rotations as well as impairment in limb use (seebelow). Bilateral models of Parkinsonism may better mimic human diseasebut they result in adipsia and aphagia in rats.

The procedure was performed in anesthetized animals (Ketamine/xylazine)via stereotactic injection in 2 sites along the median forebrain bundle.The efficiency of complete lesion induction is highly dependent on theexperience of the operator and during development of the presentinventions ranged from 60-80%. The animals were allowed to recover andthen subjected to a battery of behavioral tests starting 2 weeks aftersurgery.

ii—Core Behavioral Analysis. Behavioral analysis is initiated 2 weeksfollowing surgery and continues after transplantation until the animalis sacrificed. Its purpose is 1) to establish that the lesion is stableand complete and that the animal has not exhibited spontaneous reversalof the symptoms, a phenomenon that has been shown in partially lesionedanimals, 2) to demonstrate the impact of transplantation of dopamineneurons on established behavioral parameters.

-   -   a—Rotational Behavior. Rats are observed for spontaneous        rotations and for D-amphetamine-induced (ipsilateral) rotations        (10 mg/kg). A threshold of >6 rotations per min is required as        an indicator for a significant lesion. Apomorphine-induced        rotations can also be analyzed but positive results are        contemplated to require >80-90% depletion of dopamine        innervation in the caudate-putamen and are less consistent in        MFB lesions, in comparison to CPU lesions. Three sets of data        are obtained at 2 week intervals and averaged. Rats with a        rotation score of <6 are not included in the studies.    -   b—Stepping Test. This is a test for forelimb akinesia. A rat is        held with one hand by the experimenter fixing the hindlimbs        (slightly raising the torso) and with the other hand fixing the        forelimb that is not to be monitored. In this way the other        forepaw has to bear the weight. The rat is moved slowly sideways        in both forehand and backhand positions. The number of adjusting        steps for both directions and both paws are counted.    -   c—Cylinder Test. This is a test for forelimb use asymmetry. The        rat is placed in a transparent cylinder. During a time period of        5 min the rearing behavior of the rat is scored. The behavior is        analyzed during rearing and landing. The percentage of        simultaneous and asymmetric use of the paws during these        movements is determined. This can be carried out via a        computerized video monitoring system. Negative results on these        tests were known to correlate with dopamine depletion and        results were shown to be improved following restorative grafting        as described herein.

iii—Grafting. Animals receive stereotactic injections of dopamineneurons into the striatum, at 3 weeks following theParkinsonism-inducing lesions, and if behavioral testing confirms anadequate lesion. The coordinates for injection are widely established.Following grafting, the same set of behavioral tests is performedbimonthly for variable durations (on average 5 months are required toachieve stable behavioral recovery).

iv—Tissue Analysis. Animals are euthanized and perfused. The brains aresectioned and processed for immunohistochemistry and stereologicalanalysis. Antibodies include TH, FoxA2, Pitx3, Nurr1, Lmx1a, Girk2, DATfor dopamine neuron identity and function; 5-HT to identify Serotonergicneurons; Human NCAM or human nuclear antigen for human identity; Nestin,Sox2 for neural precursors; Ki-67 for proliferation; Oct4, Nanog forpluripotency markers; alpha-fetoprotein; myosin; cytokeratin formulti-lineage markers to rule out teratomas formation. Quantitativeparameters include graft volumes (Cavalieri estimator), total cellcounts and dopaminergic cell counts (using TH/FoxA2 double labelingneurons) and Proliferative index (% Ki67+). Antibodies listed arecommercially available and used during the development of the presentinventions.

In some embodiments, Sprague-Dawley (SD) rats are contemplated for usein order to better model the human situation. SD rats will receive dailyintraperitoneal injections of cyclosporine (15 mg/kg) starting one dayprior to grafting until sacrifice. With long term injections of theaqueous from of cyclosporine (Neoral, an oral solution used in humans)there was negligible morbidity.

In some embodiments, exemplary methods for scaling up rnDA neuroncultures are provided, see, Table 9. In particular embodiments, suchmethods are contemplated for use in producing GMP level cultures forclinical use.

Assessment Parameters. In vivo testing was contemplated for use in shortterm and long-term methods of graft effectiveness. Results obtainedduring the development of the present inventions showed tissuecharacterization identical in both cases, with an expectation ofincreased proportion of TH+ cells in long term survivors, due todifferentiation of grafted precursors.

On average when 15,000 TH+/FoxA2+ neurons per 250,000 cells weretransplanted the animals survived 5 months post grafting. Significantlyless double labeled cells survived and counted in short survivals. Thuscontemplated in vivo yields, as shown in Table 9 are conservativeestimates. Exemplary parameters used to assess a Pass/Fail status forgraft composition results are shown in Table 10.

In long-term grafts, behavioral assessment is an essential component ofthe performance of hES line products. Guidelines that define loss offunction and recovery were best described for the amphetamine rotationswhereby a robust graft should normalize the behavior and occasionallyresult in contralateral movement due to DA imbalance. The limits listedin Table 11 are exemplary guidelines as determined during thedevelopment of the present inventions.

In some embodiments, cell sources for use in differentiation methods ofthe present inventions include but are not limited to WA09, ACT (M09),Bio-Time and Roslin cell lines. In some embodiments, cell sources foruse in differentiation methods of the present inventions include but arenot limited to GMP grade lines. In some embodiments, cell sources foruse in differentiation methods of the present inventions include but arenot limited to production of mature DA neurons for use in short termengraftment survival analysis. In some embodiments, cell sources for usein differentiation methods of the present inventions include but are notlimited to production of mature DA neurons for use in engraftmentexperiments that include behavioral assessments. Controls will consistof the Research Grade WA09 and a sham saline group. Statistical analyseswill use ANOVA with the Dunnett post-hoc test.

A. Complex behavioral assays for assessment of degree of striatalreinnervation. Standard behavioral tests (as described herein) exhibiteda direct correlation with the number of surviving dopamine neuronswithin the graft. In rats treated with mature DA neuronal cells of thepresent inventions, behavior tests show maximum recovery with anestimate of about 30% DA neuron recovery which is required to reduceamphetamine-induced rotations. Thus, once a threshold for survival of DAneurons in the host is reached (an estimate of 800-1200 DA neurons inthe rat model), it may be difficult to distinguish behavioraldifferences reflecting enhancement strategies. A microtransplantationapproach was described that results in placement of multiple smallgrafts throughout the striatum, as opposed to large grafts. Whilecontrolling for the total number of DA cells transplanted, differenceswere observed in behavioral outcomes when comparing 2 groups: Rats withmultiple small grafts exhibited earlier and more extensive behavioralrecovery in drug-induced rotations and the stepping tests when comparedto animals with a single large grafi and the same number of DA neuronsand the same amount of dopamine release. Surprisingly, there was adistinct difference in forelimb skilled use, whereby animals with smallwidely distributed grafts exhibited improvement while rats bearingstandard grafts did not. Skilled forelimb use is considered a task thatrequires spatial and temporal control over DA release, and thus properconnectivity between the grafted neurons and the host striatum. Thus insome embodiments, use of mature DA neurons of the present inventions inengraftment procedures resulted in the recovery of forelimb use.Accordingly in some embodiments, mature DA neurons of the presentinventions are administered to one location of the striatum. In otherembodiments, mature DA neurons of the present inventions areadministered to at least 2 or more locations within the striatum.

B. Skilled Forelimb Use (or the Staircase) Test. The forelimb testanalyzes forelimb reaching and grasping and was performed pre- andpost-lesion and post-transplantation. Animals were food deprived for 48hours prior to the test, and tested daily for 5 days pre-lesion thentwice after lesion at 3 week interval. Following grafting, the test isrepeated 2 more times, at months 3 and 5. Animals were placed in aplexiglass chamber equipped with a double staircase. Food pellets areplaced on 5 steps bilaterally for pre-graft testing, and unilaterally(on the affected limb side, contralateral to the rotation) aftergrafting. The animals are tested over a set time frame (e.g. 10 minutes)and the numbers and ratio of pellets that were eaten (successful reach)and the pellets that were taken were calculated and compared to theindividual animal's pre-lesion performance. There were severalvariations of this test involving the number of iterations and thetiming of the test.

VIII. Differentiated Cells By Using Methods Described Herein ShowedElectrophysiology Responses Similar To DA Cells In Situ.

The following example describes using exemplary methods of the presentinventions for determining the functional capability of midbrain DAneurons resulting from differentiation by methods described herein.Substantia nigra pars compacta (SNpc)DA neurons in vivo exhibit anelectrophysiological phenotype that differentiates them from most otherneurons in the brain. In particular, they spike spontaneously at a slow(1-3 Hz) rate. Moreover, this slow spiking is accompanied by a slow,sub-threshold oscillatory potential. After 2-3 weeks in vitro, thesesame physiological features are displayed by SNpc DA neurons culturedfrom early postnatal mice. In order to determine whether the mDA neuronsof the present inventions showed a comparable electrophysiologicalphenotype, the mDA neurons of the present inventions were tested fortheir electrical response signature. Midbrain DA neurons of the presentinventions on day 80-100 of culture were tested by single cellrecording. These mDA neurons differentiated from hESCs consistently (4/4tests) displayed this distinctive physiological phenotype by showingspecific autonomous spiking behavior and oscillatory membrane potentialchanges (FIG. 3g-i ). This behavior is known as autonomous pacemakingactivity and a specific property of midbrain DA neurons and inparticular the subtype of midbrain dopamine neurons most relevant forParkinson's disease (substantia nigra type midbrain DA neurons).

Electrophysiological measurements are contemplated for use in acuteslice preparations, i.e. from biopsies of engrafted areas. In oneembodiment, A9-versus A10 type graft-derived DA neurons will beidentified in vivo based on testing for the autonomouse pacemakingactivity that is specific to A9-type dopamine neurons that are mostaffected in PD. In other words, A10 type neurons do not have pademakingactivity

Conditions were established for the in vivo recording of humanpluripotent stem cell derived DA neurons in acute slice preparations,see, FIG. 26. Specifically, grafted human DA neurons derived frompluripotent stem cells were measured for and discovered to haveelectrophysiological features typical of those seen in mouse substantianigra pars compacta (SNpc), FIG. 26A hwere the top view showsreconstruction of a pacemaking neuron in the graft region. Bottom showsan exemplary photomicrograph of a brain slice taken from the rat intowhich the hES-derived neurons were injected 9 months prior; the graft isoutlined; a higher magnification image is shown inset at the bottom. Theslice was processed for tyrosine hydroxylase which shows up as white,FIG. 26B. Further, the top view shows an exemplary cell-attached patchrecording from a putative DA neuron in the graft; Bottom shows anexemplary whole cell recording from the same cell. Recordings were madein the presence of glutamate and GABA receptor antagonists (50 μM AP5,10 μM CNQX and 10 μM GABAzine) to eliminate synaptic input. Theserecordings demonstrated that the PS-derived neurons were autonomouspacemakers with normal intrasomatic voltage trajectories. Another neuronrecorded in a graft sample had similar properties, FIG. 26C. Forcomparison, cell-attached and whole cell recordings from a dopaminergicneuron in SNpc of an adult mouse are shown. Abbreviations (CTx=cortex,STr=striatum, SNpc=substantia nigra pars compacta, DA=dopaminergic).This data shows in vivo functional studies in grafted rat striatummonths after transplantation. Thus in some embodiments, in vivofunctional studies on grafted tissue demonstrates recovery of substantianigra pars compacta (SNpc).

IX. Directed Differentiation of PINK1 Mutant Genetic PD-iPS Cells (PINK1Mutation) into DA Neurons Revealed Parkinson-Like Abnormalities in theMature DA Neurons.

This example described the discovery that large populations of midbrainDA neurons developed with characteristics of a PD patient's neurons whena PD patient's cell line, i.e. PINK1 mutant PD-iPSC cell, obtained in amanner that did not result in the destruction of an embryo, were used asthe cell population for obtaining FOXA2/LIM1XA/TH+DA neurons of thepresent inventions.

In one embodiment, the inventors' contemplate isolating a starting cellpopulation from a patient for use in the methods of making authentic DAneurons in vitro, where the patient has a symptom of Parkinson's disease(PD), for the potential advantage of using the treated cells in in vitrotests for 1) observing differentiation or functional abnormalitiescompared to authentic DA neurons from humans not having a neurologicalsymptom, then 2) using an observed abnormality for developing atherapeutic treatment for reversing that abnormality and 3) treating thepatient with the therapeutic treatment for reducing, i.e. reversing, asymptom of Parkinson's disease.

In one embodiment, the inventors' contemplate isolating a starting cellpopulation from the same patient for deriving authentic DA neurons foruse in transplantation treatment, where the patient has a symptom ofParkinson's disease (PD), for the potential advantage of reducingimmunological rejection, i.e. transplantation rejection. In otherembodiments, reduction of transplantation rejection is contemplated byusing a beginning cell source isolated from a human whose MajorHistocomaptibility Antigens (MHC) match (ie. Twin) or a human having anacceptable MHC tissue match for transplantation (such as a relative tothe patient or an unrelated human expressing overlapping MHC molecules.

A. Directed differentiation showed that genetic PD-iPS cells PINK1 cellscontained the capability to develop into midbrain like DA neurons. See,FIGS. 20-25. A PINK1 Q456X mutant PD-iPSC line was differentiated usingthe novel floor-plate based midbrain DA neuron protocol (method)described herein which yielded midbrain DA neurons that expresseddifferentiation profiles comparable to those obtained from the novelfloor-plate based midbrain DA neuron protocol differentiated 1-19 line.(FIG. 20). This example described the discovery that large populationsof midbrain DA neurons developed with characteristics of a PD patient'sneurons when a PD patient's cell line, i.e. PINK1 mutant PD-iPSC cell,obtained in a manner that did not result in the destruction of anembryo, were used as the cell population for obtainingFOXA2/LIM1XA/TH+DA neurons of the present inventions.

PINK1 Q456X mutant PD-iPSC line was differentiated using the novelfloor-plate based midbrain DA neuron protocol (method) of the presentinventions which yielded midbrain differentiation profiles comparable tothose obtained from the iPSC H9 line. A-C) Immunocytochemical analysisof PINK1 mutant PD-iPSC line at day 11 of differentiation (midbrainprecursor stage) for FOXA2 (red), LMX1A (green) and DAPI (blue) (A), day25 of differentiation (early postmitotic DA neuronal stage) for FOXA2(red) and TH (green) (B) and for NURR1 (red) and TH (green) (C). D-F)Same set of immunocytochemical analyses performed using H9 derived cellsat day 11 of differentiation for FOXA2 (red), LMX1A (green) and DAPI(blue) (D), at day 25 of differentiation for FOXA2 (red) and TH (green)(E) and for NURR1 (red) and TH (green) (F).

B. Genetic PD-iPSC expressed a PD like phenotype of protein aggregation.FIGS. 21-24. The inventors discovered that PINK1 mutant PD-iPSC showedevidence of α-synuclein (major component of Lewy body on PD patience)expression in cytosol of TH+DA neurons at day 55 of differentiationusing the novel floor-plate based midbrain DA neuron induction protocol,(FIG. 21a-b ). A, B) Immunocytochemical analysis of PINK1. mutantPD-iPSC line at day 55 of differentiation for α-synuclein (LB509, red),TH (green) and merged image (A) and α-synuclein (red) and ubiquitin(green) (B). These α-synuclein positive cells also showed highexpression of ubiquitin (classical Lewy body marker). In contrast, DAneurons derived from control iPS line showed expression of normalsynaptic (as opposed to cytosolic) α-synuclein expression and very lowlevels of Ubiquitin (FIG. 21c-d ). C, D) Immunocytochemical analysis ofcontrol-iPSC line at day 55 of differentiation for α-synuclein (red) andTH (green) (C) and α-synuclein (red) and ubiquitin (green) (D).

C. Expression of aggregated form of α-synuclein. In the PD patientbrain, dimerized insoluble form of α-synulcein leads to aggregation inLewy body. The dimerized form of α-synuclein shows phospholylation ofSerine 129 on α-synuclein. At the same day of differentiation, PINK1mutant PD-iPSC derived cells showed strong expression for Ser129phosphorylated α-synuclein in contrast to control-iPSC derived cellsthat showed very low levels of expression (FIG. 22).

PINK1 mutant PD-iPSC derived cells showed strong expression for Ser129phosphorylated α-synuclein in contrast to control-iPSC derived cellsthat showed very low levels of expression. A, B) Immunocytochemicalanalysis for Ser129 phosphorylated α-synuclein (green) and DAPI (blue)in PINK1 mutant PD-iPSC derived cells at day 55 of differentiation (A)and matched control-iPSC derived cells (B).

D. Differences in α-synuclein expression patterns are observed dependingof differentiation protocol. The inventors contemplated that floor-platederived “authentic” midbrain DA neurons showed PD specific vulnerabilityand corresponding, specific, in vitro phenotypes. DA neurons obtainedusing the classical MS5 stromal feeder based differentiation protocol(Perrier et al., PNAS 2004, herein incorporated by reference) yieldedlarge numbers of TH+ neurons. However, based on data obtained during thedevelopment of the present inventions, the inventors showed that MS5based TH+ cells were not authentic floorplate derived midbrain DAneurons. In cultures differentiated via the MS5 protocol, there weremany α-synuclein positive cells. However, those cells did not co-expressTH. Moreover, there was no difference in expression patterns betweenPD-iPSC and control-iPSC when using the MS5 differentiation strategy(FIG. 23a-b ). These data indicate that α-synuclein is also expressed inother non-DA cell types and that such non-DA α-synuclein is unchanged indisease versus control-iPSC derived cells—particularly when usingstandard MS5 differentiation protocols. These are the DA-like rosettederived neurons reported in publications (e.g. Perrier PNAS 2004). ThoseMS5 based TH+(=DA-like) cells are used for comparison in FIGS. 3, 10, 13and 16. These data indicate that α-synuclein is also expressed in othernon-DA cell types and that such non-DA α-synuclein is unchanged indisease versus control-iPSC derived cells—particularly when usingstandard MS5 differentiation protocols. Finally, the new floor platebased differentiation protocol described herein, yields large number ofTH+ cells co-expressing α-synuclein. Those TH+ cells express α-synucleinin a cytosolic expression pattern. FIG. 24A, B) Immunocytochemicalanalysis for α-synuclein (LB509, red), TH (green) of P1NK1 mutantPD-iPSC line at day 60 of MS5 based differentiation (A) and control-iPSC(B). C) Immunocytochemical analysis of PINK1 mutant PD-iPSC line at day55 of floor-plate based differentiation for α-synuclein (red), TH(green).

E. DA neurons derived from genetic PD-iPS cells are more vulnerable totoxic stimulation. FIG. 24-25. PD-iPSC derived TH+DA neurons derived viafloor-plate based protocol were more vulnerable to toxin challenge(valinomycin: mitochondria ionophore, 5 uM (ranging in concentrationfrom 1-10 uM), 48 hr) than control-iPSC derived cells. In contrast, TH+neurons derived via the classic MS5 based protocol did not showdifferential vulnerability between PD-versus control-derived cells (FIG.24). Entire cell viability assay with alamar-blue after 48 hrs ofvalinomycin treatment also showed differential cell survival in aspecific dose range for toxin challenge (5 and 10 uM) when comparingPD-iPSC and control iPSC (FIG. 25). Normal condition both of PD- andcontrol-iPSC derived cultures obtained via MS5 based protocol (D,PD-iPSC derived cells shown), TH+ neurons following toxin challenge inPD-iPSC (E), and control-iPSC derived cultures (F) obtained via MS5protocol. G-H) low power images of immunocytochemistry for Tuj1 (red)and TH (green) by floor-plate based protocol at day 60 ofdifferentiation: PD-iPSC of normal (G), versus toxin challenge (H)conditions and control iPSC of normal (I), versus toxin challenge (J)conditions. K-N) low power images of immunocytochemistry for Tuj1 (red)and TH (green) by MS5 based protocol at day 60 of differentiation:PD-iPSC of normal (K), versus toxin challenge (L) conditions and controliPSC of normal (M), versus toxin challenge (N) conditions. F.

Exemplary quantification of cell viability—dose response assay for toxinchallenge. Cell viability assay with alamar-blue after 48 hrs ofvalinomycin treatment showed differential cell survival in a specificdose range for toxin challenge (5 and 10 uM) when comparing PD-iPSC andcontrol iPSC (day 60 of floor-plate based differentiation). Note: thisassay tests for overall cell death while the most dramatic effects wereobserved specifically in DA neurons (see FIG. 14). Therefore, alamarblue based quantification will likely underestimate the extent of thedifferential effect observed on DA neuron lineages.

X. Contemplated Large Scale Culture Using Compositions and Methods ofthe Present Inventions for Providing Exemplary mDA Neurons.

The descriptions herein show exemplary methods and uses for large-scaleproduction of mDA neuronal cells resulting from differentiation bycompositions and methods described herein. The scalable generation (i.e.methods contemplated to be successful for generating mDA neuronal cellsfrom cultures containing a relatively small number of cells) were shownto yield cell populations capable of transplantable mDA neurons at Day25. In particular for PINK iPSC cells. See, Table 9).

XI. Methods to Enrich for Midbrain DA Neurons.

Several methods were developed and tested prior to and during thedevelopment of the present inventions with the goal of enriching cellpopulations for midbrain DA neuron precursors by overcoming problemssuch as by depleting contaminating cell populations, including but notlimited to contaminating pluripotent stem cells. Initial studies wereperformed using primary embryonic mouse neurons, embryonic rat neuronsand mouse ESC derived populations. In addition to having goals ofincreasing neural populations for use in DA neuron enrichment, the mouseESCs studies included developing procedures for preventing teratomafoitnation which was problematic in previous procedures. Thesestrategies included negative selection for cell surface markersexpressed on pluripotent cells (such as SSEA1) along with positiveselection of cells expressing neural marker (NCAM). Using mouse cells,several genetic reporter strategies were proposed for use in identifyingenrichment for neural cells in DA neuron transplantation paradigms (forexample, identifying cells with expression of SOX1, Corin/Lmx1a, Ngn2,TH, Pitx or DAT). Functional testing was performed in primary cells fromNgn2-reporter mice (Thomposon et al., Exp Neurol. 198(1):183-98 (2006))and from Lmx1A-reporter mice also sorted for Corin (Jonsson, Exp Neurol.219(1):341-54 (2009). For mouse ESC derived populations studies wereperformed using SOX1 (Barraud et al., Eur J Neurosci. 200522(7):1555-69), TH (Kelly et al., Minerva Endocrinol. 1991 16(4):203-6),Pitx3 (Hedlund et al., Stem Cells. 2008 26(6):1526-36) and DAT (Zhou etal., Stem Cells. 2009 27(12):2952-61) mouse ESC reporter lines.Additionally, during the development of the present inventions, theinventors performed a comprehensive transplantation study directlycomparing the in vivo performance of three purified mouse ESC-derivedpopulations representing sequential stages of DA neuron development:Midbrain precursors (Hes5::GFP), early postmitotic cells (Nurr1::GFP),and mature DA neurons (Pitx3::YFP). Those studies identifiedNurr1-expressing DA developmental as particularly suitable for graftingand demonstrated that purified DA neurons are capable of efficientengraftment in vivo. Furthermore, these results were used for selectionof cells at day 25 of differentiation (onset of Nurr1 expression) foruse in grafting hESC-DA neurons into mouse, rat and rhesus monkey modelsof PD.

During development of the present inventions production of authenticmidbrain DA neurons from hESCs showed excellent in vivo performance (seeFIG. 4), and the use of protocols described herein resulted in a DAneuron yield, at the time of grafting of approximately 40%. Thispercentage exceeded the percentages DA neurons obtained followingdissection of human fetal ventral midbrain tissue (typicallyapproximately 10%) Thus use of a hESC-based source of DA neurons asdescribed herein is contemplated for even further improvements in puritywhen using cell purification strategies. Additionally, genetic reporterlines were developed for use with hESC similar to those used in mousestudies, including a human cell line as a Nurr1::GFP line. However, theuse of genetic reporters may be problematic for translational use inhumans because GFP is immunogenic in humans thus not suitable for humanuse. Furthermore, FACS sorting may be problematic for establishingclinical grade DA neuron master cell banks (i.e. developing frozenstocks of human DA neurons for use in transplantation) given the lengthof time that would be required for sorting batches of the approximately10⁹ cells required for each transplant in addition to potentially highcosts and lower cell yield after recovery from storage.

In contrast to genetic reporter systems and FACS based cell isolation,the inventors contemplated isolation of DA neurons based on surfacemarker expression using alternative strategies for cell separationtechniques contempalted for use in PD patients. For example magneticbead sorting (e.g. CliniMACS® system) was widely used in FDA-approved,cell-based applications and allowed for rapid and cost-effectiveisolation of up to 10¹⁰ cells under GMP-compliant conditions, i.e.conditions approved for isolating cells for use in humans. Thus in someembodiments, magnetic bead sorting is contemplated for enrichment ofmature DA neurons, for example, using CD142 attached to magnetic beadsfor enriching Nurr1+ neurons for use in grafts.

XII. Identifying Cell Surface Markers For Use In Methods Of ProvidingMature DA Neurons.

Cell surface marker expression data collected during the development ofthe present inventions showed identification of several novel cellsurface markers expressed on midbrain DA neurons. Specifically, markersfor further identifying cells, such as specific cells that would matureinto DA neurons, mature DA neurons of the present inventions and A9cells, were found. Two main strategies were used to identify suchsurface markers: first, an unbiased gene expression screen in geneticreporter lines (FIG. 27a ) showed several candidate markers, includingCD142 and a marker termed DCSM1, that was selectively expressed inmidbrain DA neurons and appeared to specifically mark A9-type DA neurons(FIG. 27b . A second strategy was the use of a CD cell surface markerscreen in hESC derived DA neurons which tested for 242 commerciallyavailable antibodies in 96 well format (FIG. 27c,d ). The results ofsuch an exemplary screen (FIG. 27e ) led to the identification of atleast 5 validated markers enriched in midbrain DA neurons includingCD142, a marker that selectively marked a Nurr1+DA neuron stage (FIG.27f ), in addition to, CD63, CD99, and DCSM1.

Specifically, as illustrated in FIG. 27, a CD surface marker screen forWA09-derived DA neurons at day 25 of differentiation tested for up to242 individual antibodies. These results were compared to duplicatescreens of a broad range of other WA09 derived neural cell types (e.g.hESC-derived HB9::GFP+ motoneurons, hESC derived cortical neurons, hESCderived 1Vkx2.1::GFP+ ventral forebrain precursors, and several otherhESC derived neuron types. The resulting database of surface markerexpression profile was then used to select candidate CD markersselectively enriched in any given subtype such as midbrain DA neurons(FIG. 27). One of the markers discovered associated with hESC DA neurondifferentiation was CD142. CD142 selection of cells enrichedspecifically for hESC derived DA neurons at Nurr1+ stage while depletingother neuron subtypes. In some embodiments, CD142 is expressed beforeNurr1+. In some embodiments, a midbrain DA neuronal cell populationsorted for CD142 has Nurr1+ and Nurr1− cells. In some embodiments, amidbrain DA neuronal cell population sorted for CD142 has Nurr1− cells.In some embodiments, cultured Nurr1-CD142+ sorted midbrain DA neuronalcell population begin expressing Nurr1 (i.e. become Nurr1+) in up to twodays after sorting.

In addition to CD142, CD63 and CD99 were markers enriched on hESCderived DA neurons. Thus is some embodiments, DA neuronal cultures areenriched for DA neurons by sorting or selecting from markers includingbut not limited to CD142, CD63, CD99, DCSM1, Nurr1+, etc. CD142typically marks approximately 30% of the total cell population at day 25of differentiation (FIG. 28a ). Selectivity of CD142 for Nurr1+DA neuronstage was confirmed in multiple independent hESC and hiPSC lines (FIG.28b ). Importantly, in addition to enriching for DA neurons, CD142selectively depletes other neuron subtypes such as GABA and Serotonergicneurons. (FIG. 28c-f ). In vivo studies were performed that demonstratedthe ability of CD142 to give rise to high purity DA neuron graftswithout detectable contaminating GABA and Serotonergic neurons.Serotonergic neurons are a cell type that has been implicated in humanfetal tissue grafting as the potential source of graft-induceddyskinesias. Although grafting methods described herein using unpurifiedcells already resulted in few Serotonergic neurons, the use of CD142should further reduce this risk.

A. Markers For Identifying A9 type mature mDA Neurons. A9 derived vs. A10 derived DA neurons were found to have distinct in vitro and in vivofunctional properties and innervations patterns specific to their rolein mesostriatal versus mesolimbic function. During the development ofthe present inventions the inventors discovered that the authentic mDAneurons produced by methods of the present inventions (FIG. 4) gave riseto neurons with having more A9 than A10 characteristics. In particular,authentic mDA neurons that were TH+ at least in part expressed Girk2, amarker used to define A9 type DA neurons. Additionally many mature DAneurons exhibited autonomous pacemaking activity that is a functionalfeature present in A9 but not A10 type DA neurons. However, some TH+cells generated in vitro were not of A9 identity. Thus the inventorscontemplated enrichment procedures such as those described herein, forproviding purified populations of human A9 type authentic mDA neurons(versus A10) neurons. As described herein, the inventors discovered atleast two markers unique to A9 type neurons and at least two markers atunique to at least A10 type neurons. Thus in some embodiments, A9 typeneurons are identified by (Girk2, Aldhl) versus A10 (Calbindin, Otx2)markers.

B. Defining a marker set that enhanced yield of midbrain DA neuron withan A9 subtype. At least two strategies were contemplated for defining A9specific surface markers: Candidate markers were obtained from a geneexpression screen, such as described herein, and candidate CD-antibodiesfrom a surface marker screen, as described herein. Populations Inanother method, global transcriptome analysis in purified populations ofmouse ESC derived mDA neurons at distinct stages of differentiation(using BAC transgenic technology; see FIG. 27a,b ). Surface markers werediscovered in a surface marker profile on DA neurons derived from WA09RCB with the following exemplary methods. RCB WA09-derived DA neurons atday 25 of differentiation were dissociated and replated onto 96 wellplates, followed by exposure to the 242 CD antibodies and data analysisusing the Operetta high content scanner. Amount of DA-enrichment wastested for at least 5 additional antibodies which bound to CD markersidentified in these screens (for examples, CD142, CD63 and CD99).Candidate CD-positive versus CD-negative cells were assessed using theDA QC assays, including expression of FOXA2/TH and TH/Nurr1 (see, Table7). In some embodiments, global gene expression profiles arecontemplated for comparison of unsorted to CD142+ cells. In someembodiments, cells sorted/separated expression a desired marker wereused in short-term. and long-term in vivo studies as described herein.Among the DA neurons specific markers identified in these studies was asurface marker gene that was termed DCSM1 (DA cell surface marker 1).Based on in situ expression data, expression in the ventral midbrain butmore surprisingly found expression to be at least partially A9selective, both in the developing and adult mouse brain (FIG. 27) and inthe human adult brain. Numerous cells expressing DCSM1 expression inhESC derived DA neurons were observed. In vitro assays for A9 vs A10identity included long-term differentiation of marker+ cells (day 50 andday 75 of differentiation) and analysis of i) expression for A9, (Girk2,Aldhl) versus A10 (Calbindin, Otx2) markers in mature neurons, (ii)differential axon guidance responses to Netrin-1 and Sema3 and, (iii) A9enriched neurons were assessed by electrophysiology tests. A9 DA neuronsexhibited specific functional features as described herein for hESCderived A9 neurons. In vivo studies were performed for cells expressingDCSM1 and other markers in order to confirm i) A9 marker expression(Girk2, Aldh1) versus A10 (Calbindin, Otx2), ii) graft DA fiberoutgrowth and iii) electrophysiological A9 properties in slicepreparation of the grafted cells (see FIG. 26).

XIII. Use of Polysialic Acid (PSA) And Polysialyltransferase (PST)Enzyme.

Graft integration and extent of DA fiber outgrowth are challenges ingrafting methods including the fetal grafting studies in PD patients.One problem encountered with graft tissues and cells is limited fiberoutgrowth from these grafts when treating patients. This problem isparticularly critical in humans since patient recovery requiresextensive striatal reinnervation. In previous methods, achievingadequate reinnervation after a tissue graft required multiple injectionsof cell deposits across the striatum. Each injection can cause striataldamage and inflammation along with other surgical risk. Such risksinclude the injury of a blood vessel during cell injection that couldpotentially induce a stroke or seizures in the patient. PSA is a naturalcell surface sialic acid homopolymer (i.e. alpha 2,8-linked sialic acid)that has been identified as a posttranslational modification (throughthe action of polysialyltransferase (PST) enzyme) of other cell surfacemolecules, such as (polysialylated) neural cell adhesion molecule(NCAM), NCAM (CD56), and the like. PSA appeared to function inregulating plasticity of some cell behaviors that required changes incell-cell interactions, including cell migration and axon outgrowth.While highly expressed in the embryo, PSA was down regulated in adulttissues with the exception of localized regions of the CNS that maintainstructural and physiological plasticity (such as hippocampus,suprachiasmatic nucleus, SVZ). Thus in some embodiments, polysialic acid(PSA) was contemplated for use in promoting fiber outgrowth of engraftedcells. Examples of PSA use in other cell types are described in WO2006/042105 herein incorporated by reference in its entirety. In someembodiments, the inventors contemplate the use of authentic DA neuronsin combination with PSA as described herein.

A. Increased PSA In DA Neurons For Use In PD Patients. Major challengesremain for providing methods and cells based upon previous results fromthe use of small animal models, including limited survival oftransplanted cells and poor fiber innervation of host tissue. The impactof these limitations is contemplated to be more severe in the largerhuman striatum, and thus increased survivial and innervation isnecessary for effective clinical application of ES-derived DA neurons.Improved fiber outgrowth and graft integration in animal models,including the use of at least one, up to several injections iscontemplated to represent reductions in risk associated with multipleinjections or poor distribution of DA neurons in vivo.

Regulation of cell interactions by polysialic acid (PSA) is one of thefactors that promoted cell distribution, axon outgrowth and targetinnervation during vertebrate development, see, for example,Rutishauser, Polysialic acid in the plasticity of the developing andadult vertebrate nervous system. Nat Rev Neurosci 9, 26-35 (2008). PSAwas a carbohydrate polymer attached to the neural cell adhesion molecule(NCAM), that attenuated cell-cell interactions, and thereby promotedtissue plasticity. In a glial scar, enhanced expression of PSA in theadult brain promoted the migration of neuronal precursors from thesubventricular zone into the cortex, and improved axonal growth (El etal. Use of polysialic acid in repair of the central nervous system. ProcNatl Acad Sci USA 103, 16989-16994 (2006). As described herein,engineered increased PSA expression on purified mouse ES-derived DAneurons resulted in improved graft cell counts, extensive DA neuronfiber outgrowth into host striatum and surprisingly, enhanced behavioralrecovery in Parkinsonian mice. Further, genetic engineering ofESC-derived DA neurons for increased cell surface PSA levelsconcurrently increased in vivo survival and fiber outgrowth into hoststriatum. One exemplary embodiment is shown in FIG. 29 for using amammalian PST gene, i.e. mouse or human. Another exemplary embodimentshown in FIG. 29 shows the use of bacterial PST, i.e. PSTnm.Specifically, as described herein, increased PSA on cells used formature DA neuron based cell therapy is contemplated for use in thetreatment of PD.

B. Use Of Mouse PST For Increased PSA Expression. In vivo resultsdemonstrated increased neuritic extensions and a significant reversal ofamphetamine-induced rotations in 6-OHDA mice that received mousePST-cell modified grafts while equal numbers cells that were not PSTmodified failed to achieve the same (FIG. 33). Moreover, improved DAfiber innervation was observed to correlate with enhanced behavioraloutcome in a PD mouse model, such as when small (injection ofapproximately 50,000 cells) PSA positive cell-grafts were made theyprovided graft integration and fiber outgrowth that were approximately70% of the areas covered compared to the use of larger (100,000 or morecells) comparative grafts. A side-by-side comparision of PSA-enhancedversus control treated ES-derived DA neuron grafts showed behavioralrecovery in the PSA group (when grafts were based on transplantation of55,000 cells each) but not observed in control cells. Transplantation of100,000 ES derived DA neurons in the mouse brain showed behavioralrecovery in both PSA-treated and control-treated ES-derived DA neuronssuggesting that grafts derived from 100,000 cells are sufficient toreinnervate the mouse brain without PSA enhancement. Thus in anotherembodiment, engineered expression of PSA expression on the surface ofauthentic DA neurons is contemplated for use in procedures for treatmentof patients with PD. When PSA was induced on neuronal cells of thepresent inventions it was identical to the PSA polymer that occurrednaturally in brain cells thus unlike the use of other cell surfacemolecules for engineering therapeutic cell types, cells engineered forPSA expression are contemplated to have little antigenicity in vivo whenused on cells for engraftment procedures in humans. Moreover, high PSAlevels on engrafted cells, either endogenouse neural precursors(Battista et al., J Neurosci. 30(11):3995-4003 (2010)), Schwann cells(Ghosh et al., Glia. 60(6):979-92(2012)) and ES-derived DA neurons ofthe present inventions did not cause detectable side-effects when usedin a variety of adult rodent model. The engineered expression of PSA toeffective levels involved the action of a single polysialyltransferase(PST) enzyme whose sole product is this unique glycopolymer.Surprisingly, the amount of expressed protein and nature of theenzymatic product was remarkably constant and closely resembled the PSAfound naturally in embryonic tissues.

As described herein, engineered PSA expression is contemplated onneuronal cells for use in engraftment procedures. In particular, PSA iscontemplated for use in preparing cells for therapeutic use byovercoming problems encountered when using other types of induced cellsurface marker expression. Further, the use of PSA expression wasreproducible in protocols that cross vertebrate species (such as usingmouse PST genes expressed in human cells) because its acceptors are alsoconsistent in structure across species and are found on the majority ofcell surfaces. Engineering PST genes into hESCs to increase PSA on DAneurons. A gene encoding the human polysialyl-transferase (hPST) wasintroduced into a hESC line (WA01) using a lentiviral vector (pLenty,Invitrogen) and as described herein. Twenty selected clones wereexpanded and analyzed for PST expression. PST-expressing hESC cloneswere differentiated to ensure that PST was not silenced in DA neurons.Quantification of PSA-NCAM at different stages of differentiation (day0, 11, 25, and 50) was done using FACS analysis and immunofluorescence(Operetta). Positive clones were subjected to the suite of DA neuron QCparameters outlined in Table 7. At least 3 clones that retain high,uniform levels of PSA-NCAM during differentiation and perform well inthe QC parameters (Table 7) will advance to assessment of the neuritcoutgrowth in PST-overexpressing hESC-derived DA neurons Selected controland PST-overexpressing hESC clones were differentiated into DA neuronsusing the standard protocol described herein, followed by cell fixationand analysis at days 25 and 50. The number and length of TH-positivefibers in such cultures were quantified with the Operetta High ContentMicroscope. The Neurite Analysis module in Harmony software 3.0quantified neurite number and length, with or without PST, and the datawas statistically analyzed using a two-way ANOVA. PST-overexpressing andcontrol hESC clones that advance from in vitro studies above, weredifferentiated again into DA neurons and transplanted into a rat modelof PD. Short-term grafts (4-6 weeks) to determine survival, PSA-NCAMexpression and neurite outgrowth were done. For each clone that passedshort-term in vivo parameters were subjected to long-term graftingstudies. For those studies animals received half or a quarter of thestandard (200×10³) dose of cells. These studies were to address whetherincreased PSA leads to increased long-term survival aftertransplantation (5 months), and whether smaller DA neuron numbers arecapable of matching or outperforming the functional capacity of non-PSTgrafts transplanted at standard cell doses (FIG. 27).

In addition, complex behavioral assays sensitive to the extent ofstriatal reinnervation were monitored to further distinguish thefunctional potential of PST-versus control DA neuron grafts. The animalswere sacrificed following completion of behavioral assays, and fiberoutgrowth was quantitated using human specific antibodies NCAM and SC121and antibodies against TH (see also FIG. 29). The intensity and spreadof the hNCAM+, SC121+ and TH+ graft was measured, as well as thepercentage of human cells co-expressing DA neuron markers (TH, FOXA2)and PSA. The density of NCAM/TH+ halo of neurites emanating from thegraft were quantified at different distances. Data was compared amonggroups using a two-way ANOVA with a Bonferroni post-hoc test. Inaddition, sections were examined for qualitative changes (e.g.branching, thickness, graft distribution and shape). In addition, somegrafts will be processed for slice electrophysiological evaluation (seeFIG. 26) in terms of A9 phenotype, synapse formation with host striatum,as well as innervation by endogenous afferents.

The following example shows enhancement of polysialic acid expressionthat improved the function of ES-derived dopamine neuron grafts inParkinsonian mice.

ES cells expressing GFP under control of Nurr1 promoter (Nurr1::GFP EScells) were stably transduced with a lentiviral vector ubiquitouslyexpressing polysialyltransferase (PST). Transduced cells showed adramatic increase in PST mRNA as compared to controls (FIG. 30A).Expression of PST was observed to be sufficient for PSA synthesis onNCAM. Accordingly, PSA-NCAM expression was greatly increased inPST-modified cells at day 14 of DA neuron differentiation (FIG. 30B-E).Both the endogenous and induced cell surface PSA on ES-derived DAneurons could be removed (FIG. 30E) by a phage endoneuraminidase (endoN)that specifically cleaved PSA's unique alpha-2,8-linked sialic acidpolymers. Surprisingly, PST transduction was not observed to affectexpression of neuronal or midbrain markers in the GFP-purified DAneurons (FIG. 30F).

Other studies in 6OHDA-lesioned hemiparkinsonian mice showed thattransplantation of approximately 100,000 ES-derived DA neuron precursorsis required to produce robust functional recovery, as measured by theamphetamine-enhanced rotation test. In the present studies, sought tograft a sub-optimal number of cells in order to be able to assessaugmentation by enhanced PSA expression. In order to transplant highlyenriched DA neuron populations that are depleted for contaminatingpluripotent cells, FACS-purified cultures at day 14 of differentiationfor expression of Nurr1-driven GFP and for the absence of SSEA-1expression (FIG. 31). Without PST overexpression, a reduction of theminimally effective graft size by half (55,000 Nurr1+DA cells) failed toproduce detectable behavioral recovery. By contrast, with enhanced PSAexpression, the same number of Nurr1/PST DA neurons resulted in asignificant correction of the PD behavioral impairment (p<0.01; two-wayANOVA), with complete recovery approximately 5 weeks after surgery (FIG.32A). PSA removal prior to transplantation by incubation with endoNindicated the specificity of PSA's enhancement, in that the endoNtreatment partially reversed the functional restitution obtained withNurr1/PST (FIG. 32A).

To examine the characteristics of the grafted cells, animals wereprocessed for immunohistochemistry two months after transplantation.There was a difference in the number of surviving Nurr1+ neurons, inthat animals grafted with the PST-transduced line had on average twiceas many GFP+ cells as animals grafted with control cells (9,300+/−1,400vs. 4,230+/−1010 GFP+ cells per graft in PST versus control samplesrespectively; FIG. 32B, p<0.05, Student's t test). Furthermore,Nurr1/PST grafts also displayed higher levels of PSA expression in vivo(FIG. 32C,D). However, the proportions of cells expressing the midbrainDA markers TH and FoxA2 within the graft core were comparable for theNurr1 and Nurr1/PST cells (TH: 62.0%+/−8.0 vs. 51.3%+/−7.0 p=0.33;FoxA2: 63.2%+/−8.6 vs. 55.4%+/−2.0, p=0.3, respectively; FIG. 32E).

Neuronal processes that emerged from the Nurr1 and Nurr1/PST cellsshowed comparable levels of TH, Girk2 (G-protein-coupled, inwardlyrectifying potassium channel) and synapsin (FIG. 33A). Unlike otherstudies with transplanted Schwann cells (Ghosh, et al. Glia 60, 979-992(2012)), enhanced PSA expression had little effect on migration of DAcells from the grafting site. However, there were clear changes inneurite outgrowth. As shown in FIG. 33B, there were more DA neuronalprocesses emerging from Nurr1/PST cells as compared to Nurr1+ controls.When the intensity of GFP and TH immunofluorescence was quantified infive successive 100 μm zones away from the transplant, Nurr1/PST graftsdisplayed a much higher relative density of processes (FIG. 33C,D;p<0.01 for both GFP and TH, two-way ANOVA). In quantifying this effect,normalized the relative density of processes to the density observed inthe most proximal zone immediate to the graft core. Such normalizationwas required to compensate for the larger number of surviving cells inthe Nurr1/PST grafts and to confirm a specific effect of PSA on neuriteoutgrowth. Specificity was also demonstrated when cell surface PSA wasremoved by endoN treatment prior to grafting. Thus pre-treatment withendoN reduced distal fiber outgrowth back to control levels (FIG. 33E).

These discoveries showed that at least some of the effects of PSA ongraft function resulted from enhanced fiber innervation of striatum.Accordingly, there was a strong correlation between graft function andthe relative extent of GFP-positive fiber outgrowth for example intozone IV (FIG. 33F; p<0.001, r2=0.65, n=17). Surprisingly, the fiberoutgrowth/behavioral relationship was consistent for experimental groups(control, PSA enhanced, and endoN-treated), indicating that graft-hostinnervation was a parameter for behavior recovery in the mouseParkinsonian model. Several factors contributed mechanistically toincreased fiber outgrowth, such as enhanced penetration of the zone ofreactive glia encapsulating the graft core, increased sprouting ability,improved outgrowth into the surrounding host tissue (e.g. easier growthcone translocation), and prevention of premature connections with hosttissue in proximity to the graft core. The exemplary mechanisms areconsistent with PSA's role in facilitating process outgrowth duringnormal development and in the adult nervous system.

The experiments described herein demonstrated the use of engineered PSAin DA neuron grafting which provided superior results compared to graftsfrom other types of cells. Data clearly indicated that PSA enhancementprovided a significant augmentation of the ability of grafted DA neuronsto innervate host striatum and attenuate PD functional deficits.Therefore clinical translation is contemplated comprising DA neurons ofthe present inventions for providing cells prior to transplantation. Insome embodiments, the cells will be genetically manipulated forexpressing PSA. In some embodiments, PST may be delivered directly tothe cells via exposure to the purified enzyme and substrate, in vitro,prior to transplantation. In some embodiments, PSA strategy for humantranslation in PD grafting is contemplated to minimize the need formultiple injections and thereby reduce the surgical risks resulting fromthese multiple injections.

In other embodiments, this technology is contemplated for use on othercell types and species, for example, augmenting the migration of graftedSchwann cells in creating a bridge (for example, cell-cellcommunication) for re-growth of axons at the site of spinal cord injury.

The following are exemplary materials and methods used in this example.

Animals: Six-week old 129S3/SvImJ mice (Jackson Laboratory) were keptunder controlled temperature with food and water available ad libitum.Experimental procedures were performed according to NIH andinstitutional animal use guidelines and approved by the localInstitutional Animal Care and Use Committee (IACUC) and theInstitutional Biosafety Committee (IBC).

6OHDA injection and amphetamine-induced test: Animals were anesthetizedwith sodium pentobarbital (10 mg/kg) and injected in the right striatumwith 2 μl of 6OHDA (4 μg/μl in saline, 0.5% ascorbic acid). Theinjections were performed with a Hamilton syringe at coordinates: 0.5 mmposterior, 1.8 mm lateral relative to bregma and 2.5 mm ventral to brainsurface. Before the surgery animals received a single i.p. injection ofdesipramine (25 mg/Kg, Sigma). Two weeks after surgery animals werescored in the amphetamine-induced rotation test. They were placed on 30cm diameter clear plastic cylinders for half an hour after which theyreceived a single i.p. injection of amphetamine (10 mg/Kg, Sigma). After20 min, the number of ipsilateral/contralateral rotations was scoredduring another 20 min. Animals were scored once a week for seven weeksthen deeply anesthetized and perfused through the heart with PBS and 4%paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains wereremoved and postfixed overnight at 4° C. in 4% paraformaldehyde thenvibratome sliced (Pelco-101, Ted Pella) in 40 μm-thick sagittalsections.

Cell differentiation and transplantation: A Nurr1::GFP BAC transgenicBAC mouse ES reporter cell line (i.e., GFP expression is driven by Nurr1promoter) 5 was transduced with a lentivirus (pLenti, Invitrogen)containing the mouse PST gene under control of the CMV promoter. EScells were propagated on mitomycin C-treated MEFs (StemCellTechnologies) in DMEM (Invitrogen), 10% FBS (HyClone) supplemented with1,400 units/ml LIF (ESGRO; Invitrogen), 2 mM L-glutamine, 1 mMβ-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin(Invitrogen). DA differentiation was induced according to Barberi etal., Nat Biotechnol 21, 1200-1207 (2003), with modifications. Briefly,cells were differentiated on MS5 feeder cells in gelatin-coated dishes(10,000 cells/10 cm dish) and cultured for four days on serumreplacement media (SRM). At day 4, Sonic hedgehog (SHH, 200 ng/ml) andFGF8 (100 ng/ml) were added. At day 7 of differentiation, the media waschanged to N2 supplemented with SHH, FGF8 and bFGF (10 ng/ml). At day11, terminal differentiation was induced by withdrawal of SHH, FGF8 andbFGF and the addition of ascorbic acid (AA, 200 μM) and BDNF (20 ng/ml).

Cells were harvested at day 14-15 with accutase treatment for 45 min,washed once with N2 and incubated with AlexaFluor-647 conjugatedanti-SSEA-1 antibody (BD Pharmingen) for 25 min. Cells were washed oncewith N2, resuspended in HEPES buffer with 0.1% BSA. DAPI was added toassess viability. FACS was performed with a MoFlo cell sorter and thepopulation of interest was sorted for GFP fluorescence (Nurr1). Thepopulation positive for AlexaFluor-647 (SSEA-1) was negatively sorted.For GFP negative control, naïve J1 mouse ES-cells were used at the samedifferentiation stage.

Nurr1::GFP sorted cells were analyzed for viability and resuspended inN2 with BDN and AA to a final concentration of 55,000 cells/μl. One μlwas injected into the lesioned mouse striatum with a 50 μm tipped fineglass capillary at coordinates: 0.3 mm posterior, 1.5 mm lateral frombregma and 2.2 mm ventral to the brain surface. An aliquot of the cellsuspension was re-plated in matrigel-coated 6 mm dishes for furthercharacterization.

For immunofluorescence analysis, cells were fixed with paraformaldehydefor 10 min at 4 0 C, washed twice with PBS, blocked with 5% BSA (0.1%Triton X-100 in PBS) and incubated with primary antibodies for 2 hrs atroom temperature: rabbit anti-GFP (1:1000, Invitrogen), mouse IgManti-PSA (1:2000, 5A5), mouse anti-NeuN (1:800, Chemicon), mouse anti-TH(1:1000, Sigma), goat anti-FoxA2 (1:800, Santa Cruz), goatanti-Engrailed (1:800, Santa Cruz). Cells were then incubated withCy-conjugated secondary antibodies (1:1000, Jackson).

EndoN treatment: To remove PSA from NCAM, the night before harvesting,cells were treated with 20 units of endoN, a phage enzyme thatspecifically removes PSA 7-9. Cells were then harvested and injected asdescribed before but were resuspended in N2 with BDNF and AA and 5 unitsof endoN. We previously assessed that the injection of the same amountof endoN alone into lesioned mice did not improve animal behavior.

PST mRNA and PSA-NCAM analysis in vitro: For Western blot analysis,cells were treated with WB buffer (PBS with 1% NP40, 150 mM NaCl, 1 mMEDTA, and 1× protease/phosphataseinhibitors added immediately beforeextraction, at pH of 7.4) and sonicated twice for 5 sec, centrifuged andresuspended in Laemli buffer (LB). Aliquots without LB were saved forprotein determination. Equal amounts of protein were loaded into 6%sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (BioRad).Proteins were transferred by electrophoresis onto polyvinylidenemembranes (Millipore). The membranes were blocked for 1-6 hr in 0.1%Triton X-100 TBS (TBS-T) with 5% non-fat dry milk and incubatedovernight with anti-NCAM antibody (1:10,000, Santa Cruz) in TBS-T with5% milk. Blots were then incubated with peroxidase-conjugated secondaryantibody (1:10,000, Jackson) and detected with ECL detection method(Amersham Pharmacia Biotech). Protein levels were quantified usingImageJ software.

For qRT-PCR analysis, total RNA was extracted with Trizol (Sigma),reverse-transcribed (Qiagen) and amplified with 10 μl of 2×SYBR reactionmixture and 0.2 μM of forward and reverse primers to a final volume of20 μl. For PSA-NCAM FACS analysis, cells were harvested with accutasetreatment for 45 min, washed once and incubated with mouse IgM anti-PSA(1:250, 5A5) for 25 min on ice, washed once with N2 media and incubatedwith Cy3-conjugated anti-mouse-IgM (1:250, Jackson) for another 25 minon ice. Cells were washed once with N2 and resuspended with 0.1% BSAwith 7AAD and analyzed in a FACS Calibur cell sorter. As control, noprimary antibody was added.

Immunohistological and stereological procedures: Free floating coronalsections were blocked in 0.1% Triton X-100, 5% donkey serum in PBS for30 min at room temperature and incubated 48 hrs at 4° C. with differentantibodies: rabbit anti-GFP (1:300), chicken anti-GFP (1:200, Chemicon),mouse anti-TH (1:200), mouse IgM anti-PSA (1:1000), mouse anti-NeuN(1:400), goat anti-FoxA2 (1:300), rabbit anti-Girk2 (1:300, AlomoneLabs), mouse anti-synapsin (1:200, BD Transduction Laboratories).Sections were then washed and incubated with secondary antibodies: Cy2,Cy3 and Cy5-conjugated donkey antibodies (1:400, Jackson). For PSA aCy5-conjugated donkey anti-IgM was used (1:500 Jackson). Incubationswere performed for 2 hrs at room temperature. Sections were washed twicein PBS and mounted in Mowiol (Calbiochem). One-in-three coronal sectionsof the brain were analyzed for each immunolabeling. Digital images werecollected by a Zeiss LSM 510 laser scanning confocal microscope withthree lasers (Argon 488, HeNe 543 and HeNe 633) with a c-Apochromat 40×objective (water-immersion). The number GFP+ and TH+ cells was countedin one-in-three sections encompassing the whole brain under a 40×objective, and the total number of cells/graft estimated. Double-labeledcells were analyzed in single optical planes through the entire z-axis.

For the analysis of the percentage of GFP/TH+ and GFP/FoxA2+ labeledcells, 100 GFP+ cells were analyzed for each marker. For processoutgrowth analysis, confocal z-scans were performed at 0.8 μm intervalsthrough the entire z-axis (20-40 μm) with a pinhole of 1 μm under a 40×objective. Sections were scanned from the injection site laterally untilno processes were observed. 3-D projections encompassing the wholescanned area were sequentially matched. For GFP and TH intensityanalysis, the entire scanned area was divided into five successive 100μm zones away from the transplant and the intensities were measuredusing ImageJ software. Data were normalized to the intensity in the zonenearest the graft (zone I) to control for any potential differences ingraft size.

Statistical analysis: Data are presented as the mean±standard error ofthe mean (SEM). Comparisons were performed using Student's t test ortwo-way analysis of variance (ANOVA) followed by Bonferroni post-hoctest. Linear regression analysis was performed and quantified using thePearson correlation.

C. Over-Expression Of The Human PST Gene (Polysialyl Transferase).

Lesioned animal groups received one of 3 doses of wild type cells orcells expressing PST or pretreated with PST enzyme. They were processedfor behavioral testing following the paradigms discussed in Table 11(amphetamine rotations, cylinder test, stepping test). The doses chosen(for examples, 200 k, 100 k, 50 k cells) were used successfully in micesuch that resulting neurons showed results as described herein for mousePST gesnes.

D. Direct Administration of Purified PST Enzyme To Cells. In otherwords, a nontransgenic method of PSA induction. Pre-treatment of cellswith the PST enzyme which results in polysialylation and increasedexpression of surface PSA. Although mammalian PSTs are low abundancemembrane proteins that operate in the Golgi, the purified Neisseriameningitides a2,8-polysialyltransferase (PSTnm) operated in anextracellular environment when using a commercially available non-toxicsubstrate (i.e. CMP-sialic acid) and produced a polymer chemicallyidentical to mammalian PSA. As one example, an active fragment of thisenzyme was effective for adding PSA to therapeutic proteins in vitro toaugment in vivo pharmacokinetics. In the presence of CMP sialic acid,PSA was synthesized by PSTnm directly on surfaces of a wide variety ofcell types in vitro, including mouse and human ESCs (FIG. 35A-E). Thedirect injection of PSTnm and substrate in vivo triggers increased PSAaccumulation on cell surfaces in adult brain regions, including cerebralcortex, striatum and spinal cord (FIG. 35GH). PSA produced by PSTnm wasdegraded by endoN (FIG. 35B) which removed induced PSA, demonstratingthat PSTmn-produced PSA has functional properties comparable toendogenous PSA (FIG. 35A,C, D). PSA expression via PSTnm occurred inless than an hour which overcame the slow PST transgene induction.Expression persisted for several weeks in vivo after which PSA markerswere reduced thus overcoming side effects from prolonged induction of aPST transgene. Therefore the use of in vitro cell exposure toPSTnm+substrate is contemplated as a simple, alternative strategy fortriggering increased PSA levels in hESC derived DA neurons and othercell types for use in engraftment procedures. In part, this alternativeapproach for translation, i.e. use in human engraftment procedures, hasthe advantages of being non-invasive nature, i.e. avoiding the use oftransgenic methods, GMP-grade reagents are used in these procedures formeeting clinical use protocols, and having a transient nature ofbiochemically-generated PSA expression matching the expected time framefor DA fibers leaving the graft core and entering the host brain,necessary for avoiding some dangerous side effects of using cellsengineered for engraftment.

The following example shows enzymatic engineering of PSA on hESC-derivedDA neurons using the purified bacterial polysialyltransferase, PSTnm, toenhance transplant efficacy.

Although effective, PST gene transfection necessitated geneticmodifications of hESCs with limited control over the duration ofpolysialylation. This exemple describes the discovery that externalPSTnm induced PSA, instead of gene delivery, (see, FIG. 35). In FIG.35A, PST treated Schwann cells (SC) (green line-middle line) hadincreased adhesion time while PSTnm-produced PSA inhibited adhesion. Inparticular, (A) PSTnm-produced PSA inhibits adhesion of Schwann cells insuspension to a Schwann cell monolayer even more effectively (redline-lowest line) than PSA produced by forced PST expression (greenline-middle line). (B) PSA immunoblotting in ESC-derived HB9 motoneuronsshows that control samples treated with PSTnm alone had undetectablelevels of PSA. Incubation with PSTnm+CMP-sialic acid substrate producesa large PSA band, which is removed with endoN treatment. (C, D) Similarto effects obtained with the PST gene, polysialylation of these cells byPSTnm and substrate during differentiation enhances neurite outgrowthand cell migration (arrowheads). (E) PSA immunostaining of day-30hESC-derived DA neurons. (F) This staining is significantly increasedafter treatment with PSTnm and substrate. (G) In vivo injection of PSTnmalone has no effect, while its co-administration with substrate (H)produces large amounts of PSA expression in mouse striatum.

Thus mature DA neurons externally treated with PSTnm is contemplated foruse in the producing cells for engraftment. Both mammalian PST and PSTnmproduced chemically identical chains of PSA. Increased PSA onhESC-derived DA neurons (FIG. 35F) should persist for several weeks,sufficient for DA fibers to exit graft core. Because PSTnm is removedprior to grafting, immunogenicity to this enzyme contaminating thegrafted cells should not be factor.

PSTnm was produced from an engineered fragment with enhanced solubilityand activity characteristics (Willis et al., Characterization of thealpha-2,8-polysialyltransferase from Neisseria meningitidis withsynthetic acceptors, and the development of a self-primingpolysialyltransferase fusion enzyme. Glycobiology 18, 177-186 (2008)).Cultures of hESC were induced to differentiate into DA neurons beforePSTnm exposure, exposure to substrate or both. Cultures were examined atdifferent time-points of exposure (10 min to 6 hrs) by quantitativeimmunofluorescence (Operetta) and western blotting to determine thespeed and levels of polysialylation. Thus, Day 25 differentiatedhESC-derived DA neurons will be incubated with the optimumconcentrations of PSTnm and substrate using the conditions describedherein. PSA+mDA neurons will be transplanted in short- and long-termassays as described herein and in FIG. 29.

E. Applications For Spinal Cord Injury. Several studies aimed towardsthe eventual use in human patients and strategies described herein,showed broad potential of these procedures including PST-gene deliveryby injecting lentiviral vectors expressing PST directly into the CNS topromote axon regeneration and endogenous progenitor migration. One suchstrategy for use in human procedures targets spinal cord injury. In onetype of spinal cord regeneration procedure, Schwann cell grafts wereused as part of the therapy for rebuilding cellular bridges for use inin vivo axon regeneration. However, patient recovery of locomotorfunction, including fine muscle control, resisted any therapeuticintervention. As shown herein, enhancing PSA expression on Schwann cellsused for engraftment resulted in enhancement of Schwann cell migrationand axonal growth which further resulted in dramatic effects onincreasing locomotor function (FIG. 30A-D). Thus in some embodiments,increasing PSA expression on Schwann cells in vitro is contemplated foruse in engraftment procedures in humans having spinal cord injury.Another strategy for use of engineered expression of PSA by increasedexpression of PST genes in human procedures involved HB9 ESC-derivedmotoneurons, where introduction of the PST gene in these neurons vialentiviral vector based gene expressionresulted in a dramatic increasein the outgrowth of axons both in culture and after grafting in mice forrepairing a mechanically induced sciatic nerve injury. The latterresulted in an improved and specific targeting of muscle tissue (FIG.30E-H). Thus in another embodiment, engineered expression of PSAexpression on the surface of ESC-derived motoneurons for use inrestoring function of the sciatic nerve.

IVX. Increased Safety Of DA Neuron Grafts.

Contemplated embodiments for further reducing health risks to patientsfor use in methods of producing cells of the present inventions aredescribed below.

Although the cells produced by methods described herein demonstratedcharacteristics for reduced risks to patients when used for engraftment,additional embodiments are contemplated to further reduce thepossibility of risk to the health of patients receiving engrafted cells.One of several concerns for hESC-based cell therapy procedures is thepossibility of introducing contaminating undifferentiated cells thatresisted differentiation which under post engraftment conditions developinto cells that cause harm to the patient. In the case of a pluripotentcell, one harmful result is teratoma formation that endangers thepatient's life. Teratoma formation using hESC derived cells was reportedfollowing short-term neural differentiation protocols based onspontaneous cell differentiation. However, the inventors' use of humanES derived neural cell types, unlike their mouse ESC-derivedcounterparts, rarely resulted in teratoma formation followingappropriate neural differentiation strategies as described in thecurrent invention (i.e. monolayer culture, dual-SMAD-inhibition protocoland growth in cytokines that do not promote proliferation). In factafter analyzing several hundred animals with human cell grafts, using avariety of neural differentiation strategies over the last 10 years,teratoma formation was not observed. Furthermore, teratomas were notobserved in PD transplantation procedures of the present inventionsusing human cells for grafts. The difference between the use of humanvs. mouse cells for engraftment procedures for use in humans iscontemplated to be related to the different stage of pluripotencycaptured in human versus mouse ESCs, whereby human cells are thought tomatch the properties of a pluripotent stage described as Epi-SCs, unlikemouse ESCs which may be at a different developmental stage.

However, at least one of the problems using cells from previoustransplantation studies was the continued risk of neural overgrowth thatis substantial in Perrier et al., PNAS 2004 and similar protocols whichare surprisingly absent when using mature DA neurons and transplantationmethods of the present inventions. Further, another problem found inengraftment tests in previous studies was the formation of hESC derivedneuroepithelial structures (i.e. neural rosette-type) that continue toproliferate in vivo. This in vivo expansion of grafted neuroepithelialcells was observed in various neural transplantation paradigms includinghESC derived DA neuron transplantation studies in rodent Parkinson's andHuntington's disease models. Those “neural rosette-type” proliferatingcells represented non-transformed primary cells with a high intrinsicgrowth potential which resulted in large grafts composed of ectopic,mostly cortical-type tissue in grafted animals. As described herein,several strategies were used to eliminate contaminating pluripotent orneuroepithelial cells at the time of grafting (e.g. selection for SSEA-4(pluripotent marker) or Forse-1 (neuroepithelial marker). Thesestrategies were partially successful when using rosette-baseddifferentiation strategics and neural overgrowth was still observed in asubset of grafts sorted for Forsel or sorted negatively for S SEA-4.Surprisingly, with the development of this floor plate-based rather thanrosette-based DA neuron differentiation procedures, the issue ofneuroepithelial overgrowth was overcome. Rarely observed weregraft-derived, proliferating cells within functional hESC floorplate-derived DA neuron grafts.

Based upon the adverse results of other engraftment procedures, anothersafety concern for using DA neuronal grafting into humans are sideeffects of the therapy such as the occurrence of graft-induceddyskinesia (GID) observed in about 15% of patients receiving fetaltissue in transplantation trials. However, as discussed herein, thenearly complete absence of graft derived Serotonergic cells, along withthe use of a more consistent cell source with the possibility of furtherdepleting unwanted cell types (see, text associated with FIG. 28), andthe possibility of controlling in vivo DA fiber distribution (see, FIG.29; i.e. preventing “hot spots” of neuronal clusters secreting L-Dopaand other compounds) are major advantages of using the methods of thepresent inventions over other methods for providing cells for use inengraftment procedures. Thus use of the methods of the presentinventions is contemplated to minimize risk to patients.

VX. Use Of Human ESCs For Clinical Translation.

in preferred embodiments, human ESCs are contemplated for use in methodsfor making and using cells for engraftment procedures in humans, inother words, cell therapy for the treatment of PD.In particular, humanESCs have numerous advantages over using human iPSCs in methods of thepresent inventions, such as, for one example, for use in providingengraftable midbrain DA neurons for use as a PD cell therapy. Inparticular, the use of induced pluripotent stem cells (iPSCs) as a cellsource for DA neuron derivation has several advantages, such asproviding a genetically matched cell source for each patient. However, anumber of recent studies have created uncertainty regarding the safetyand full genetic compatibility of iPSCs. Reprogrammed cells have beenshown to harbor potentially dangerous genetic and epigeneticabnormalities that are undesirable for clinical utility. Furthermore,work in mouse iPSCs showed that iPSC derived cells are not fullyimmunocompatible which is the main argument to support their use inhuman transplantation. Furthermore, there are no FDA-approved proceduresusing iPSCs for engraftment. Finally, it would be impractical andcost-prohibitive to envisage the generation of GMP-compliant and fullyQC-controlled cell banks for each individual patient. In comparison, thegenetic stability of hiPSCs compared to hESCs was intensively studied.Although hESCs were observed to acquire mutations over time in culturethe timing and rate of such mutations appeared to differ from hiPSCs,where hESCs were generally considered more genetically stable thaniPSCs, for examples, see, Hussein, et al., Nature 471, 58-62 (2011);Mayshar, et al. Cell Stem Cell 7, 521-531 (2010); Lister, et al. Nature471, 68-73 (2011); Laurent, et al. Cell Stem Cell 8, 106-118 (2011).Additionally, standard operating procedures were devised for severalhESC lines that satisfied the rigorous safety tests required by the FDAfor cellular and gene therapy products. The FDA has approved two groupsin the United States to advance hESC-based cellular therapies toclinical use. For example, Geron Corporation entered a Phase I trialwith hESC-derived oligodendrocyte precursor cells (GRNOPC1), andAdvanced Cell Technology (ACT), Inc. has two current Phase I/II trialsusing hESC-derived retinal pigmented epithelial cells to treatStargardt's Macular Dystrophy (trial NCT01345006) and Advanced Dry AgeRelated Macular Degeneration (trial NCT01344993).

Finally, the fact that both of the FDA-approved, hESC-based clinicaltrials target nervous system disorders show advantages of usinghESC-based methods for providing engraftment material for treatingnervous system diseases and injuries. The nervous system is consideredan immuno-privileged site since foreign tissue (allografts) elicits weakimmune responses when compared to the same graft placed into theperiphery. In fact, after twenty-five years of transplanting fetal cellsinto human brains, it was found that some allogenic neurons survived forup to 16 years in the human brain with transient immunosuppression.Therefore it appears that identical antigenic matching between a cellsource and the graft recipient is not essential. Thus hESCs arecontemplated as a universal, allogenic source of DA neurons for treatingPD in addition to other nervous system diseases, disorders and injuries.Patients receiving cells of the present inventions are contemplated tohave a clinical diagnosis of PD. Authentic DA neurons grafts arecontemplated for use in early intervention and moderate-to-severe PD,including patients in whom there is insufficient symptomatic controlwith available medications, such as levodopa, adjunctive medication,etc. In some embodiments, patients contemplated to receive neuron graftshave subtle signs in early PD (e.g. by the use of neuroimaging fordetecting dopaminergic deficits, FDG-PET, and signs of dyskinesias, etc.Dyskinesia as measured by the Unified Dyskinesia Rating scale (UDysRS)(Goetz, et al., Mov Disord. 23, 2398-2403 (2008)) is contemplated foruse in monitoring patients before and after engraftment. Patients mayalso have “scans without evidence of dopaminergic deficits” (SWEDDS),some of whom may have dystonia or essential tremor. Brain MRI would bedone in order to identify patients with other (non dopa) contributoryfactors to Parkinsonism. In some embodiments, patients would have apositive response to levodopa. Determining pre- and post-transplantationparameters and endpoints for subject monitoring, such as motorevaluation, non-motor evaluation, quality of life, and also the use ofneuroimaging and other biomarkers. Motor function: The UPDRS andnewly-validated MDS-UPDRS (Goetz, et al., Mov Disord. 23, 2129-2170(2008)) are widely used for measuring PD motor symptoms. However, othertests including 10 m walk or 6 minute walk tests, timed up and go,functional gait assessment, functional reach, and others arecontemplated for more patient-oriented outcome measures. Patients wouldbe tested in the “off” state, as well as “on”, and rating scales wouldbe included for “off” time (for example following Movement DisorderSociety recommendations based upon clinimetric properties of validatedwearing off scales (Antonini, et al. Mov Disord. 26, 2169-2175 (2011))and dyskinesia rating scales (for example UdysRS (Goetz, et al., MovDisord. 23, 2398-2403 (2008)). Videotaping standardized patientexaminations in both “on” and “off” states is contemplated. Non-motorfunction: Measures will primarily target cognitive, psychiatric outcomesand dysautonomia in addition to addressing cognition, depression,anxiety, apathy, sleep, fatigue, psychosis, and other non-motor symptomsbefore and after engraftment. Quality of life: PD-specificquestionnaires (PD-QUALIF) and/or well-validated quality of life scalessuch as the SF-36 are contemplated to monitor patient outcomes.

Neuroimaging and other biomarkers: Functional imaging was widely used insurgical PD trials. While dopamine-based imaging (such as FDOPA-PET) iscontemplated for use in examining graft maintenance, neuroimagingtechniques using other ligands are contemplated for use includingimaging-based markers, for example targeting inflammation, and ofnon-imaging systemic markers as exploratory data collection. Imagingwould find use in pre-operative planning, e.g. extent and location of DAdepletion within the basal ganglia and incorporation of PET data intailoring surgical planning for each patient. Location of graftplacement and number of cell deposits. In some embodiments, the putamen(Freed, et al. N. Engl. J. Med. 344, 710-719 (2001); Lindvall, et al.Prog. Brain Res. 82, 729-734 (1990) and postcommissural putamen (Olanow,et al. Ann. Neurol. 54, 403-414 (2003)) are sites of cell engraftment(administration). In some embodiments, multiple placements via differentsurgical tracks are contemplated. MRI with a Clearpoint system whichprovides real time imaging and visualization of the trajectory path andtargeting accuracy is contemplated for monitoring the engrafted cells.This system is used in placement of Deep Brain Stimulation electrodes inPD patients. Cell number and Composition of the graft. Fetal trials wereperformed with essentially unknown number of DA neurons since they usedfetal graft material with numerous cell types. Based upon data providedherein, an estimated 100, 000-200,000 surviving TH+ neurons arecontemplated for recovery of DA neuronal function.

Immunosuppression. In some embodiments, immunosuppression of graftedpatients is contemplated, at least 6 months up to the lifetime of apatient. In some embodiments, patients will not be immunosuppressed forpurposes of having engrafted tissue.

TABLE 1 Gene expression array data of significantly up-regulated anddown- regulated genes at differentiation day 11 in SHH/FGF8/Chir treatedFloor-plate based population over control LSB treated population. ColumnColumn Fold- Column Column Fold- # ID Change p-value # ID Change p-value7304 FOXA1 22.5512 5.03E-16 12640 IRX3 3.32454 1.65E−05 7305 FOXA217.3328 5.70E−17 26837 OSBPL10 3.3051 9.55E−07 31270 SPON1 16.43932.14E−14 32086 THBS4 3.29135 9.77E−07 31684 SYT4 13.2693 9.61E−12 14581LOC100130506 3.1293 5.81E−09 4334 COL22A1 8.54042 2.05E−17 4931 DAB23.02851 4.80E−09 6616 FBLN1 8.24162 4.97E−18 12642 IRX5 2.95938 2.73E−0832035 TFF3 8.01805 1.08E−13 13774 LMX1A 2.81057 1.42E−06 5324 DKK17.95664 1.19E−09 31516 STOM 2.76317 2.01E−07 3244 CAPN6 7.58928 1.77E−0826374 ODZ4 2.75644 3.06E−06 2544 C20ORF56 7.58575 6.71E−15 8210 GSC2.75053 3.47E−07 27591 PKDCC 7.42271 2.02E−11 26170 NRCAM 2.712641.82E−08 23584 LOC91461 5.43556 9.86E−09 28414 PTCH1 2.63379 2.99E−085037 DDC 5.19301 2.09E−08 31578 SULF2 2.59351 1.42E−07 13598 LDB24.76303 2.92E−09 7366 FREM1 2.5697 2.25E−07 723 AMOT 4.58838 5.68E−0813743 LITAF 2.55681 3.65E−08 4978 DBX1 4.40946 2.36E−09 26236 NTNG12.55383 1.78E−07 31138 SOX8 4.32227 9.29E−08 25869 NEUROG2 2.550125.05E−06 30193 SILV 4.3198 2.52E−07 26132 NPY 2.54575 4.48E−06 30148SHISA2 4.31109 1.60E−07 25967 NKX2−1 2.52195 0.00023 31509 STK39 3.656242.90E−06 27101 PCDH18 2.46481 1.69E−05 27661 PLCL2 3.63607 6.81E−1132557 TNFRSF19 2.45297 4.06E−08 24425 MGST1 3.59223 2.48E−11 5759 EFEMP12.44308 7.78E−09 32945 TSPAN7 3.57361 7.36E−07 31148 SP5 2.434595.34E−06 29082 RHOU 3.51768 5.89E−06 28069 PQLC3 2.42685 2.59E−05 9283HS.19193 3.49663 6.24E−13 30313 SLC20A2 2.40457 1.24E−06 5478 DOCK103.43476 8.18E−07 30743 SNHG4 2.35277 8.69E−08 25094 MMP11 3.407052.13E−08 27607 PKP2 2.34734 5.34E−05 29975 SERPINF1 3.36169 1.97E−1025520 MYL4 2.3327 3.93E−08 29042 RGS4 3.33544 7.01E−06 26128 NPTX22.29568 1.08E−05 926 APCDD1 3.33059 3.47E−10 26006 NMD3 2.29039 3.18E−0519975 LOC646966 2.26444 6.23E−05 16924 LOC391019 2.28656 1.25E−05 29894SEMA6A 2.25036 0.000844 29352 RPL15 2.05205 2.82E−06 6161 EYA2 2.231750.00024 33800 WNT5A 2.03874 1.08E−06 26105 NPFFR2 2.23095 7.31E−05 30481SLC38A4 2.03653 1.13E−06 4818 CXXC4 2.23047 1.66E−05 12059 HS.70932.01993 2.66E−06 2764 C4ORF14 2.22128 5.73E−06 22500 LOC728126 2.01831.16E−06 18269 LOC642989 2.22053 0.000735 16095 LOC136143 2.017755.71E−05 13059 KIAA1324L 2.21906 4.75E−05 12718 ITPR3 2.01384 1.67E−0523663 LRIG3 2.21159 4.38E−07 4004 CHN2 2.00649 7.18E−06 6196 FABP5L22.19875 3.09E−05 32162 TIPARP 2.00538 0.000633 29347 RPL13A 2.193620.000122 482 ADSS 2.00282 0.000136 12446 IL1RAPL1 2.19134 1.75E−05 2196C17ORF45 2.00241 6.15E−05 28455 PTN 2.18577 9.36E−05 3447 CCDC51−2.00054 0.000177 30746 SNHG7 2.18224 4.35E−07 5945 ENC1 −2.00380.000468 28788 RASL12 2.16856 1.39E−05 27915 POU3F2 −2.00877 2.04E−0717150 LOC401074 2.16782 1.07E−06 23143 LOC729779 −2.01527 0.000972 30283SLC16A3 2.16541 6.06E−05 23972 MAP1LC3A −2.01892 3.37E−06 15378LOC100133008 2.16461 3.15E−05 7611 GAS6 −2.02463 9.63E−08 32180 TLE62.16433 6.35E−07 17062 LOC399959 −2.02793 2.39E−05 12887 KCNQ1OT1 2.16412.97E−06 29519 RSPO3 −2.03762 1.61E−05 31210 SPATS2L 2.16393 0.00059729038 RGS20 −2.04076 3.60E−09 4241 CMTM8 2.16297 0.000392 19157LOC644936 −2.04097 0.000128 30933 SNORD25 2.15953 0.000363 30502 SLC3A2−2.04467 5.58E−05 2803 C5ORF13 2.15734 3.94E−05 33508 VCAM1 −2.054212.51E−06 32131 TIGA1 2.15335 1.80E−05 490 AFAP1L2 −2.05673 8.23E−07 1667BMP7 2.14281 0.000497 23275 LOC730167 −2.06009 0.000652 20837 LOC6499462.14098 8.39E−05 29215 RNF175 −2.06406 6.81E−05 25756 NCRNA00219 2.125436.42E−07 5923 EMILIN2 −2.06713 2.81E−06 8003 GPM6B 2.08967 3.45E−0534367 ZNF462 −2.07292 1.63E−05 5650 DYM 2.08942 0.000179 26244 NUAK1−2.0732 0.000172 29606 S1PR3 2.08344 0.000479 25818 NEBL −2.073511.55E−06 27681 PLEKHA5 2.08285 0.000217 29624 SALL3 −2.0787 2.15E−0611495 HS.570308 2.08044 1.67E−06 33491 VANGL2 −2.08497 0.000402 26232NTN1 2.06755 2.49E−08 30581 SLC7A8 −2.08722 1.40E−08 12635 IRS1 2.064350.000109 875 ANXA3 −2.08727 0.000227 20404 LOC648343 2.05497 0.00079134075 ZFP36L1 −2.08803 5.08E−05 1227 ASNS −2.10169 8.03E−05 15637LOC100133760 −2.09319 8.14E−05 33804 WNT7B −2.1052 3.00E−08 33087 TUBB2A−2.36013 0.000777 24064 MARS −2.107 0.000146 31747 TAGLN −2.368092.16E−05 33725 WDR72 −2.11201 0.000617 5372 DMRTA1 −2.36884 5.28E−0831295 SPRY1 −2.12339 2.43E−05 3614 CD200R1 −2.37345 1.25E−14 7495GABARAPL1 −2.12367 1.70E−05 17147 LOC401056 −2.37854 8.57E−10 4331COL1A2 −2.12521 1.97E−05 33801 WNT5B −2.3794 3.01E−08 6821 FILIP1−2.1288 0.00026 28590 RAB11FIP1 −2.40833 6.65E−07 24246 METRN −2.131181.99E−05 23362 LOC730525 −2.41129 1.59E−06 12056 HS.66187 −2.137892.97E−05 27658 PLCH1 −2.4212 3.31E−07 28438 PTGIS −2.14246 7.36E−06 7000FLJ32310 −2.42179 5.77E−06 12980 KIAA0367 −2.15304 1.92E−05 3339 CBX4−2.43314 1.13E−07 1575 BCMO1 −2.16946 7.40E−05 6073 ERRFI1 −2.445391.44E−05 25473 MXRA5 −2.17542 3.63E−06 32521 TMSB15A −2.45505 1.31E−0532537 TNFAIP1 −2.17984 7.66E−06 13242 KLHL14 −2.51767 2.71E−11 34064ZFHX4 −2.23036 0.00046 30201 SIPA1L2 −2.52018 3.67E−06 28787 RASL11B−2.23056 0.000115 32760 TRIB1 −2.52274 1.73E−06 5632 DUSP6 −2.233660.000732 13641 LFNG −2.53204 5.33E−07 3149 CACHD1 −2.23514 5.39E−0512285 IER3 −2.54594 1.65E−07 3221 CAMKV −2.25983 5.01E−08 28506 PTPRZ1−2.55447 1.62E−06 7248 FLNC −2.26856 1.13E−05 1608 BEX2 −2.563310.000109 28307 PRSS8 −2.28456 1.35E−05 32179 TLE4 −2.56513 0.00013512662 ISYNA1 −2.29255 1.40E−05 10888 HS.551307 −2.56708 0.000405 7524GADD45A −2.2962 7.46E−07 683 ALPK2 −2.57443 2.83E−06 5921 EMID2 −2.299951.55E−05 32551 TNFRSF12A −2.59274 4.02E−06 941 APLP1 −2.31453 2.03E−0732610 TNRC9 −2.59782 3.23E−07 30576 SLC7A5 −2.32091 0.000757 777 ANKRD1−2.61554 3.15E−05 7474 FZD3 −2.32644 6.13E−06 13622 LEMD1 −2.623512.97E−05 5940 EMX2OS −2.3303 2.65E−07 26777 OR7E156P −2.64884 3.51E−071259 ATF3 −2.34034 2.59E−06 24377 MGC39900 −2.66479 1.46E−05 33806 WNT8B−2.34155 3.96E−08 12267 ID1 −2.66562 6.68E−07 31388 SST −2.346631.83E−06 27726 PLP1 −2.69279 9.13E−07 3188 CALCB −2.34833 3.49E−06 4350COL4A6 −2.69704 1.17E−07 1075 ARHGEF6 −2.35421 1.54E−08 4320 COL11A1−2.71039 1.79E−09 26885 OTX1 −2.35607 0.00023 3127 CA2 −2.71276 2.35E−0826393 OLFM3 −2.35759 2.32E−07 26151 NR2E1 −2.72059 4.25E−14 13744 LIX1−2.73818 9.34E−06 31267 SPOCK1 −2.7283 3.38E−05 32038 TFPI −2.776856.23E−05 3067 C9ORF171 −3.24146 3.73E−11 32776 TRIM24 −2.79151 8.73E−0812363 IGFBP3 −3.34999 1.02E−06 32756 TRH −2.79963 3.98E−07 4533 CRIP1−3.45501 6.62E−08 32652 TOX3 −2.81075 9.35E−06 3740 CDH11 −3.486092.23E−08 2405 C1ORF21 −2.81457 9.49E−09 816 ANKRD38 −3.48622 6.93E−125371 DMRT3 −2.82614 4.28E−09 12730 JAG1 −3.58463 3.83E−08 32381 TMEM2−2.83489 1.83E−05 12271 ID4 −3.72402 1.78E−12 2437 C1ORF61 −2.853243.53E−09 810 ANKRD34B −3.76606 1.15E−11 2868 C6ORF141 −2.8569 6.05E−0612850 KCNJ13 −3.81092 2.16E−09 1178 ARX −2.87861 0.000122 6112 ETV5−3.81591 4.37E−10 26245 NUAK2 −2.95666 4.14E−08 9265 HS.181245 −3.866111.24E−09 33796 WNT2B −2.96258 1.15E−10 633 ALDH2 −3.87108 1.97E−09 28507PTRF −2.97439 2.07E−07 26180 NRIP3 −3.92066 6.08E−08 7662 GCNT1 −2.981081.49E−06 30413 SLC2A1 −4.11899 1.18E−05 5930 EMP1 −2.9918 1.11E−06 5479DOCK11 −4.35251 5.67E−09 31844 TBC1D9 −3.00324 2.53E−09 25658 NAV1−4.35259 1.02E−10 4352 COL5A2 −3.02084 1.26E−07 5349 DLL1 −4.474741.58E−07 4699 CTNNA2 −3.04046 6.91E−09 34418 ZNF533 −4.49025 1.16E−092516 C20ORF177 −3.04277 4.86E−08 327 ACTC1 −4.63509 7.37E−09 3518 CCL2−3.04332 9.50E−11 1037 ARHGAP15 −4.67561 9.89E−10 34315 ZNF385B −3.050845.56E−07 31132 SOX3 −4.72958 7.45E−09 12058 HS.7023 −3.0642 3.72E−0826153 NR2F1 −4.73849 1.53E−09 23661 LRIG1 −3.07681 3.30E−07 3129 CA4−4.7904 3.00E−09 7813 GLI3 −3.07936 1.22E−08 33071 TTYH1 −4.790458.94E−14 31513 STMN2 −3.09098 4.93E−06 5766 EFHD1 −4.86615 1.16E−0930023 SFRP1 −3.09686 1.13E−06 867 ANXA1 −4.95401 1.41E−08 1128 ARMCX2−3.09746 2.91E−06 8500 HES4 −5.24189 1.63E−11 4689 CTGF −3.114164.47E−08 4299 CNTNAP2 −6.14001 2.94E−08 6365 FAM181A −3.1422 2.56E−0923947 MAMDC2 −6.36522 2.23E−10 4121 CLDN1 −3.15492 1.93E−06 7605 GAS1−6.38074 2.63E−11 627 ALDH1A1 −3.15745 1.18E−12 31151 SP8 −8.889654.42E−20 629 ALDH1A3 −3.17887 4.46E−13 30024 SFRP2 −9.73482 4.24E−1433501 VAT1L −3.18253 1.21E−06 13677 LHX2 −10.9076 6.16E−11 4912 CYR61−3.18856 3.58E−07 5939 EMX2 −11.5085 1.34E−19 12781 KANK4 −3.200547.99E−12 21068 LOC650757 −12.1496 7.71E−20 5554 DRD4 −17.3929 3.17E−1627068 PAX6 −15.7636 2.24E−22 26065 NOS2A −20.0139 2.83E−16 8501 HESS−38.4549 8.90E−22 1501 BARHL1 −20.6077 2.59E−14 26064 NOS2 −25.17517.45E−17

TABLE 2 Gene expression array data of significantly up-regulated anddown- regulated genes at differentiation day 11 in SHH/FGF8/Chir treatedFloor-plate based population over SHH/FGF8 only treated population.Column Probeset Fold- Column Probeset Fold- # ID Change p-value # IDChange p-value 31575 SYT4 14.8775 3.24E−12 31926 TFF3 3.62059 1.12E−087195 FOXA1 11.8929 2.65E−13 25244 MSX1 3.60243 0.000146141 4225 COL22A18.26361 3.21E−17 23475 LOC91461 3.58231 2.08E−06 5215 DKK1 6.395071.32E−08 26777 OTX2 3.47814 3.97E−09 7956 GPR177 5.98201 5.65E−12 13489LDB2 3.44124 3.22E−07 13508 LEF1 5.07514 2.45E−11 13657 LMO3 3.291972.61E−12 2435 C20ORF56 5.02955 2.71E−12 817 APCDD1 3.21277 6.95E−10 4928DDC 5.0147 3.23E−08 4822 DAB2 3.18324 1.84E−09 11952 HS.71947 4.810985.13E−09 25760 NEUROG2 3.16851 1.30E−07 13665 LMX1A 4.7485 3.59E−1031101 SPATS2L 3.15744 2.80E−06 12427 INHBE 4.72434 3.41E−07 1311 AXIN23.13274 1.80E−06 32562 TPBG 4.63162 1.44E−09 32448 TNFRSF19 3.111042.48E−10 1152 ATF5 4.48583 0.000153048 4934 DDIT3 3.08248 4.83E−05 5369DOCK10 4.23427 4.29E−08 26776 OTX1 3.05293 6.14E−06 31414 STOX1 4.206953.94E−10 28360 PTPN13 3.03567 5.18E−06 4869 DBX1 4.17519 5.31E−09 994ARL4A 2.98803 1.45E−07 31977 THBS4 4.13195 3.66E−08 26728 OSBPL102.94508 5.19E−06 2694 C5ORF13 4.0095 7.84E−10 32836 TSPAN7 2.860941.65E−05 31469 SULF2 3.83814 8.51E−11 5665 EFNB2 2.78085 0.00022256331468 SULF1 3.81974 3.96E−07 8101 GSC 2.75066 3.46E−07 34285 ZNF5033.81092 2.76E−08 6426 FAM84B 2.74213 7.89E−07 12531 IRX3 3.735983.93E−06 29866 SERPINF1 2.73192 1.26E−08 4935 DDIT4 3.73152 3.78E−05 924ARHGAP10 2.72522 3.36E−07 31369 STC2 3.70632 8.48E−08 31039 SP5 2.681629.57E−07 27482 PKDCC 3.69823 2.20E−07 28210 PSAT1 2.64735 9.82E−06 4257COLEC12 3.6503 5.20E−06 28933 RGS4 2.6312 0.000148375 28424 PVRL32.55083 1.04E−07 13547 LGI1 2.61567 1.30E−06 299 ADAMTS9 2.531312.88E−08 7369 FZD7 2.61404 2.46E−06 1555 BMP4 2.52708 9.40E−05 25351MUSTN1 2.55841 4.85E−07 31161 SPON1 2.5133 0.000145864 25411 MYL42.55345 4.77E−09 26265 ODZ4 2.50312 1.46E−05 5851 ENPP2 2.100480.000485738 33730 XBP1 2.50164 1.72E−06 1558 BMP7 2.0876 0.00071857328766 RBP1 2.49237 6.12E−05 23514 LPAR4 2.08062 1.96E−06 12533 IRX52.48929 7.43E−07 23554 LRIG3 2.07935 1.81E−06 1118 ASNS 2.47416 4.50E−065964 ERRFI1 2.07249 0.000220066 2770 C6ORF160 2.40219 3.58E−05 3623CDCA7 2.07177 0.000280326 12337 IL1RAPL1 2.40145 3.15E−06 30635 SNHG52.0594 4.95E−06 12526 IRS1 2.3994 7.70E−06 32620 TRAM2 2.047840.000111169 7221 FOXJ1 2.39492 8.89E−05 12809 KCTD6 2.04602 0.00011494528647 RARB 2.37013 9.11E−09 28901 RGL1 2.04043 0.000167278 14472LOC100130506 2.36331 1.36E−06 30628 SNHG1 2.03706 0.000252445 3979CITED2 2.36201 2.92E−05 27947 PPPDE1 2.02017 2.69E−05 31430 STT3B2.34069 1.29E−05 5434 DPYSL3 −2.00257 0.000139976 26023 NPY 2.33461.98E−05 23863 MAP1LC3A −2.00377 4.01E−06 6052 EYA2 2.3192 0.00013691524226 MGC18216 −2.01184 0.000204988 26991 PCDH17 2.31776 8.71E−05 4818D45234E −2.01244 8.77E−05 26127 NTNG1 2.31615 1.30E−06 4825 DACH2−2.01277 8.71E−07 373 ADSS 2.29099 1.20E−05 4386 CPXM2 −2.02106 2.02E−056120 FAM107A 2.26256 0.000677009 32224 TMEM169 −2.02651 0.00022201928387 PTPRM 2.25934 1.41E−05 151 ACBD7 −2.03074 6.94E−05 31572 SYT172.257 7.10E−05 8643 HOOK1 −2.03984 1.85E−05 7143 FLRT2 2.24042 1.35E−074278 COPG2IT1 −2.04004 8.62E−07 32372 TMEM88 2.2394 4.55E−05 32981 TUBB3−2.04044 0.000989413 27552 PLCL2 2.23488 1.24E−06 5887 EPHA1 −2.050821.41E−06 5681 EGLN3 2.23059 0.000841858 28929 RGS20 −2.06283 2.60E−093837 CHAC1 2.22744 2.61E−05 28068 PRKCH −2.06869 4.13E−06 26045 NR2F22.21836 1.71E−05 33539 WBP2 −2.0692 0.000157614 7196 FOXA2 2.216918.01E−05 23711 LY6E −2.07026 8.93E−05 12950 KIAA1324L 2.18683 6.09E−0529803 SEPP1 −2.08034 2.48E−05 3667 CDK6 2.1717 0.00076066 12713 KCND2−2.08768 1.63E−08 9540 HS.36053 2.15618 5.44E−07 34309 ZNF533 −2.088710.000231468 19012 LOC644860 2.15508 7.00E−06 14009 LOC100129034 −2.091064.54E−07 4366 CPNE8 2.15418 9.80E−06 25915 NMU −2.09244 0.000184692 4936DDIT4L 2.14147 0.000195501 24132 MEST −2.09269 2.92E−06 29691 SDCBP2.13721 3.10E−05 2715 C5ORF41 −2.09563 1.42E−05 7144 FLRT3 2.119690.00033768 27778 PON2 −2.09758 1.79E−05 6694 FHDC1 −2.11553 8.86E−0624105 MEG3 −2.10214 5.69E−07 24175 MFNG −2.11579 2.95E−05 31812 TCF7L2−2.10261 0.000153698 6640 FEZ1 −2.11631 2.58E−06 33783 YBX2 −2.11186.90E−06 2407 C20ORF177 −2.12232 3.62E−05 28329 PTGIS −2.11465 9.61E−0626783 OVOL2 −2.12627 3.53E−07 12672 KANK4 −2.29735 1.73E−08 28883 RFTN2−2.13498 7.25E−08 3505 CD200R1 −2.30795 3.06E−14 19038 LOC644919−2.13675 2.95E−06 33264 UPK2 −2.30847 1.37E−10 30004 SH3BP4 −2.137850.000314453 24978 MMD −2.317 1.46E−05 31020 SOX2 −2.14642 0.0001071696002 ETV4 −2.32842 6.04E−09 16268 LOC284422 −2.16056 1.07E−05 27655PMP22 −2.33585 4.94E−12 4819 DAAM1 −2.16701 3.85E−05 5657 EFHD1 −2.339977.47E−05 32404 TMPRSS2 −2.1693 6.26E−07 31186 SPRY1 −2.34859 3.56E−0626668 OR7E156P −2.17193 1.53E−05 3631 CDH11 −2.35006 1.77E−05 12609ITPR3 −2.18226 3.09E−06 29555 SAT1 −2.35602 2.21E−05 6372 FAM65B−2.18271 1.37E−06 33268 UPP1 −2.36162 5.38E−08 12635 JARID2 −2.185414.46E−05 1968 C14ORF4 −2.36739 2.59E−06 25024 MOBKL2B −2.19737 5.45E−059512 HS.34447 −2.37168 7.49E−08 31567 SYT13 −2.19832 2.30E−05 16243LOC283953 −2.38022 4.57E−07 25658 NDRG1 −2.20101 5.25E−07 3203 CAV2−2.38208 1.09E−11 24318 MGST3 −2.20252 3.24E−07 26223 NYNRIN −2.403111.90E−05 2147 C18ORF26 −2.2117 2.55E−06 22623 LOC728715 −2.413530.000504831 31010 SOSTDC1 −2.21381 2.23E−08 25084 MPPED2 −2.420246.42E−10 4662 CXCL14 −2.21955 1.81E−08 13403 LAMC2 −2.42245 5.32E−1033161 UCA1 −2.21966 2.19E−05 25083 MPPED1 −2.42332 3.73E−14 12176 IER3−2.21992 2.75E−06 13402 LAMC1 −2.43845 7.40E−05 7171 FNBP1 −2.231041.98E−05 15722 LOC100134265 −2.44777 4.60E−06 12199 IFITM3 −2.231674.22E−06 5836 ENC1 −2.45175 1.84E−05 25635 NCRNA00153 −2.24173 2.01E−05244 ACVR2A −2.46307 1.87E−06 27531 PLAC9 −2.24346 9.11E−08 33616 WDR72−2.46656 6.61E−05 25743 NES −2.2469 3.16E−06 28509 RAB31 −2.471991.57E−06 31405 STMN3 −2.25856 0.000796798 3629 CDH1 −2.4799 3.05E−067536 GCA −2.27697 1.34E−05 28905 RGMA −2.50113 4.45E−06 12275 IG5F3−2.28535 4.08E−06 29475 S100A11 −2.50568 3.24E−06 17038 LOC401056−2.2854 2.45E−09 8259 HAPLN1 −2.50809 7.14E−07 4533 CSRP2 −2.288990.000244001 3942 CHST7 −2.51075 9.78E−08 31606 TACSTD1 −2.289330.000115538 12543 ISL1 −2.51716 2.76E−10 29372 RRAGD −2.292580.000537359 28305 PTCH1 −2.52418 7.32E−08 5884 EPCAM −2.29475 0.0009928532541 TOX −2.5288 5.34E−09 28398 PTRF −2.29476 1.92E−05 6067 F2RL1−2.54309 2.36E−08 5241 DLL3 −2.5911 8.77E−08 32334 TMEM54 −2.577012.04E−06 33424 VGF −2.59836 0.000406676 3436 CCND1 −2.58061 6.72E−0523253 LOC730525 −2.61609 3.32E−07 707 ANKRD38 −2.58621 4.12E−09 31368STC1 −2.63136 1.20E−05 13092 KIT −3.13441 8.27E−12 31910 TFAP2C −2.637734.72E−08 6684 FGFR3 −3.13726 7.73E−09 25012 MN1 −2.6469 8.33E−08 8646HOPX −3.15424 1.07E−14 28419 PVALB −2.67012 1.23E−10 29497 S1PR3−3.15587 8.43E−07 6960 FLJ37644 −2.67066 1.78E−12 33399 VCAM1 −3.159522.02E−10 7417 GADD45G −2.68804 2.14E−06 5240 DLL1 −3.18075 1.28E−05 4709CXXC4 −2.68958 5.39E−07 28112 PRODH −3.18595 2.33E−07 8050 GRHL3−2.73097 4.15E−11 23200 LOC730278 −3.27115 1.92E−07 23990 MB1P −2.739959.29E−07 1202 ATP1B2 −3.29446 2.34E−08 5418 DPPA4 −2.75778 5.66E−08 6720FJX1 −3.3039 6.85E−07 32212 TMEM158 −2.77503 9.30E−06 3609 CDC42EP4−3.33348 2.25E−09 5896 EPHB1 −2.77518 2.29E−06 12198 IFITM2 −3.352373.41E−11 31404 STMN2 −2.79651 2.03E−05 3679 CDKN1C −3.37225 1.15E−0912454 INSM1 −2.81526 1.15E−09 3202 CAV1 −3.37517 5.73E−13 13142 KLHL24−2.81638 2.31E−07 31171 SPRED1 −3.42954 3.76E−08 9856 HS.475334 −2.823041.09E−05 30010 SH3GL3 −3.43813 3.73E−11 33125 UBL3 −2.85607 2.77E−088335 HDC −3.45787 1.51E−12 13655 LMO1 −2.90621 4.10E−06 8391 HES4−3.49495 1.00E−08 6003 ETV5 −2.90987 5.54E−08 33335 USP44 −3.503036.22E−11 966 ARHGEF6 −2.92751 9.82E−11 1101 ASCL1 −3.54534 1.26E−1131187 SPRY2 −2.94814 6.74E−09 29945 SFTA3 −3.5611 1.59E−07 28501 RAB25−2.97139 5.77E−07 13619 LIPA −3.56979 1.34E−08 4675 CXCR7 −2.98646.13E−07 27178 PDPN −3.72978 5.33E−12 32652 TR1B2 −2.99455 8.47E−08 7370FZD8 −3.74383 7.14E−09 28397 PTPRZ1 −3.02099 8.00E−08 3647 CDH3 −3.806873.54E−09 31158 SPOCK1 −3.02198 8.12E−06 2759 C6ORF141 −3.94476 5.10E−0825849 NKD2 −3.02555 4.12E−11 13595 LIMCH1 −3.94759 2.52E−11 25471 MYT1−3.03712 6.21E−09 13513 LEMD1 −3.98954 6.68E−08 6856 FLJ25404 −3.038131.95E−08 5961 ERP27 −4.02755 8.38E−13 30304 SLC2A1 −3.05478 0.00027553429666 SCRG1 −4.03799 1.96E−17 12256 IGFBP5 −3.06686 2.00E−06 7286 FRZB−4.04927 2.19E−05 23779 MAF −3.07491 2.81E−08 12977 KIAA1598 −4.049964.12E−09 28198 PRSS8 −3.08336 6.31E−08 2380 C20ORF100 −4.16273 2.94E−129923 HS.509165 −3.09539 2.57E−07 25866 NKX6-2 −4.17171 1.46E−10 4243COL5A2 −3.10066 8.01E−08 31030 SOX9 −4.21749 9.69E−10 3112 CAMKV−3.10668 3.37E−11 27805 POU3F1 −4.22404 1.53E−11 29915 SFRP2 −3.130513.42E−07 7879 GPC4 −4.23918 8.64E−08 8401 HEY1 −4.68961 2.52E−12 17977LOC642590 −4.36113 1.29E−10 30592 SMS −4.74656 5.18E−12 19608 LOC646347−4.53146 1.77E−08 29914 SFRP1 −4.84227 1.53E−09 5238 DLK1 −4.628098.12E−05 13376 L1TD1 −4.85832 6.96E−08 6675 FGF8 −7.58196 1.32E−18 7984GPR56 −4.87971 1.88E−13 31023 SOX3 −7.63996 1.53E−11 7367 FZD5 −5.002472.43E−11 8395 HESX1 −8.28818 3.36E−10 25858 NKX2-1 −5.06868 2.84E−0828692 RAX −8.87763 6.24E−12 7801 GNG8 −5.39693 2.22E−13 20959 LOC650757−9.74543 1.18E−18 9156 HS.181245 −5.99308 1.43E−12 14470 LOC100130502−10.333 1.53E−10 32962 TTYH1 −6.33607 1.00E−15 13568 LHX2 −14.16384.81E−12 5523 DUSP6 −6.34711 8.15E−10 30111 SIX3 −15.019 8.24E−13 6091FABP7 −6.56016 2.28E−09 12197 IFITM1 −16.4704 9.16E−16 5248 DLX5−6.56829 8.74E−13 25859 NKX2-2 −18.6642 7.93E−20 12754 KCNK12 −6.9121.07E−12 12254 IGFBP3 −19.1294 8.81E−16 30165 SLC15A3 −7.17663 1.60E−198392 HES5 −33.7356 2.69E−21 30114 SIX6 −39.4411 2.59E−18

TABLE 3 Gene expression array data of significantly up-regulated anddown- regulated genes at differentiation day 25 over day 13 inSHH/FGF8/Chir treated Floor-plate based population. Column Column Fold-Column Column Fold- # ID Change p-value # ID Change p-value 1209 ASCL133.085 1.60E−09 28590 PTPRO 8.70512 3.13E−10 3978 CHGA 28.1727 4.44E−1330386 SLC17A6 8.22904 2.02E−06 31604 STMN2 20.9978 1.47E−08 31767 SYT138.1735 3.63E−07 32159 TH 15.2965 2.12E−08 30390 SLC18A1 8.15448 6.47E−1531606 STMN4 15.2102 3.72E−09 5689 EBF1 8.05344 2.30E−06 7685 GAP4312.5729 5.42E−09 5029 DCX 8.00552 8.30E−08 4926 D4S234E 10.7914 4.91E−082470 C1QL1 7.47022 8.68E−10 8593 HES6 10.6527 2.94E−09 12609 INA 6.921215.72E−07 4342 COL3A1 10.0606 3.86E−06 26250 NR4A2 6.75723 1.27E−13 12149HS.7023 9.60731 3.97E−08 29624 RTN1 6.6247 6.52E−07 5691 EBF3 9.287715.13E−11 9374 HS.19193 6.58382 1.95E−08 29820 SCG2 9.22584 1.02E−06 436ADCYAP1 6.49154 9.75E−11 12655 INSM2 9.01409 3.73E−12 1607 BEX2 6.339868.93E−09 27988 POSTN 8.79262 1.09E−10 34098 ZCCHC12 6.19663 9.95E−077997 GNG3 6.1196 1.26E−06 7056 FLJ25404 6.19296 2.14E−06 25922 NEFM6.11347 2.51E−04 31605 STMN3 6.17605 8.77E−07 12359 ID2 6.07844 1.32E−069428 HS.204481 4.29602 1.66E−09 29017 REEP1 6.04393 2.25E−06 8288 GRM84.17846 1.62E−06 30810 SNAP25 6.03677 3.62E−06 7617 GADD45G 4.176632.21E−06 25671 MYT1 5.86419 8.19E−06 4298 CNTNAP2 4.13552 4.10E−05 33297UBE2J1 5.76887 6.22E−08 1408 AUTS2 4.13111 2.79E−05 5349 DLL3 5.764514.92E−08 23822 LRRC4C 4.11703 1.27E−09 29133 RGS4 5.69761 1.87E−06 31454SRRM4 4.11574 8.04E−07 28123 PPP2R2B 5.56067 2.80E−08 4003 CHN2 4.095893.30E−07 27287 PCSK1N 5.43156 3.22E−09 27665 PITX2 4.08382 1.94E−1132126 TFF3 5.26964 3.17E−08 13296 KLC1 4.0528 4.19E−06 27288 PCSK25.22877 9.14E−10 27773 PLEKHA6 4.0464 2.57E−07 4533 CRIP2 5.183758.40E−08 25618 MYLIP 3.99268 2.96E−06 12506 IL13RA2 5.1246 1.31E−06 5036DDC 3.96553 8.46E−05 32417 TMEM163 5.01765 7.55E−09 33181 TUBB3 3.939221.52E−04 30813 SNAP91 4.92061 4.28E−05 24522 MIAT 3.93906 9.87E−05 13260KIF5C 4.86241 2.93E−06 27411 PEG10 3.9374 3.48E−05 15798 LOC1001339234.83074 3.74E−07 32343 TMEFF2 3.92112 6.49E−07 32412 TMEM158 4.766464.81E−06 29821 SCG3 3.92108 6.47E−06 3770 CDK5R1 4.75524 1.18E−05 31759SYP 3.89869 2.51E−04 5739 EEF1A2 4.75232 9.53E−05 9425 HS.202577 3.890532.85E−07 32697 TNRC4 4.75179 1.29E−05 33943 XKR4 3.87043 1.49E−06 5455DNER 4.71709 1.52E−05 33178 TUBB2A 3.86265 6.11E−05 6700 FAT3 4.687851.91E−06 12654 INSM1 3.84646 5.59E−05 6197 ETS2 4.67343 1.08E−10 13236KIF1A 3.84543 3.27E−05 25753 NBEA 4.65265 6.99E−07 25721 NAPB 3.819953.50E−05 12944 KCNJ16 4.53077 3.54E−12 31765 SYT11 3.77827 1.14E−05 3979CHGB 4.52218 7.76E−05 5877 ELAVL3 3.70028 9.69E−05 25562 MXD4 4.4457.25E−07 13858 LMO4 3.69359 2.09E−06 3941 CGNL1 4.44195 7.24E−06 4103CLASP2 3.64468 1.79E−05 26499 ONECUT2 4.37403 2.39E−07 2956 C7ORF413.63616 5.69E−05 2867 C6ORF141 4.35252 2.58E−10 33362 UCHL1 3.610035.64E−05 12362 ID4 4.32183 6.05E−05 29645 RUNDC3A 3.54448 8.42E−05 26011NHLH2 4.31928 5.91E−05 24063 MAP1LC3A 3.53736 4.36E−06 2035 C14ORF1324.31666 9.46E−07 5944 ENC1 3.51677 0.000141473 7469 FRMD4A 3.461817.40E−06 28730 RAB40B 3.48084 4.57E−07 13070 KIAA0363 3.43032 6.39E−0525162 MLLT11 3.4633 3.13E−05 13342 KLHL24 3.41191 3.34E−05 3851 CELSR32.9545 6.27E−04 7988 GNB3 3.38669 3.31E−09 3177 CADM1 2.95335 1.81E−0431840 TAGLN3 3.36355 1.67E−03 20895 LOC649841 2.94392 2.21E−05 12147HS.66187 3.34592 0.000125172 13713 LEMD1 2.93152 9.17E−06 4549 CRMP13.32015 2.42E−04 8184 GPR56 2.93095 3.08E−04 23453 LOC730525 3.315011.64E−03 31935 TBC1D9 2.92862 6.60E−05 31514 ST6GALNAC5 3.29449 1.19E−058046 GOLSYN 2.9099 2.80E−06 27054 PAFAH1B1 3.25816 1.30E−04 33004 TSHZ12.90158 2.76E−07 32432 TMEM170B 3.25165 2.44E−05 5876 ELAVL2 2.901150.00136166 27686 PKIA 3.24255 4.29E−05 12733 IRX5 2.88659 1.21E−04 1748BSN 3.2101 0.000169219 1254 ATCAY 2.86583 7.41E−04 6213 EVL 3.176823.36E−05 31763 SYT1 2.86449 1.02E−03 34217 ZMIZ1 3.16249 3.29E−04 29193RIMBP2 2.84834 6.29E−06 16419 LOC283514 3.15057 4.57E−06 28741 RAB6B2.84821 0.000698361 32081 TERF2IP 3.13818 3.43E−05 27533 PHF21B 2.84576.44E−05 4932 DACH1 3.11457 7.79E−06 24135 MAPT 2.83633 5.22E−04 5424DNAJC19 3.09946 1.64E−09 10116 HS.505676 2.83499 1.50E−05 8093 GPM6A3.07619 1.59E−03 8712 HIST2H2BE 2.82687 0.000157254 15034 LOC1001317183.0686 4.01E−05 24061 MAP1B 2.82235 0.000357426 8706 HIST1H4K 3.063941.84E−06 13054 KIAA0182 2.8162 2.66E−04 28238 PRICKLE2 3.06341 1.98E−0634044 ZBTB20 2.81539 8.77E−08 31429 SRGAP3 3.0575 7.84E−06 29509 RPRM2.81168 0.000475144 13760 LHFP 3.04812 0.000126565 34007 YPEL5 2.810611.16E−04 3181 CADPS 3.03806 6.60E−06 13609 LANCL2 2.80004 8.07E−06 3186CALCA 3.03372 3.13E−08 940 APLP1 2.77078 2.57E−05 1672 BMPR2 3.031719.26E−06 29849 SCN3B 2.76654 7.14E−05 25861 NDRG4 3.00851 0.00023986829201 RIMS3 2.76205 2.94E−04 2265 C18ORF8 2.99462 9.07E−06 27394 PDZD42.75594 0.000212095 4698 CTNNA2 2.962 6.07E−05 1364 ATP6V1G2 2.753660.0012022 1606 BEX1 2.96088 5.37E−05 31778 SYT7 2.74946 4.15E−05 32769TPH1 2.96087 2.88E−07 32351 TMEM106A 2.73965 0.000217998 31986 TCEAL72.95947 3.43E−05 7717 GATS 2.72521 3.10E−05 3322 CBLN2 2.95729 1.12E−04506 AGAP3 2.71696 2.07E−05 29639 RUFY3 2.95504 2.59E−05 24141 MARCH42.70659 8.96E−04 3196 CALM1 2.68129 8.50E−05 5348 DLL1 2.7028 2.05E−0431386 SPRY1 2.67788 1.15E−03 6212 EVI5L 2.6886 6.79E−05 33128 TTR2.66999 2.76E−05 4916 CYTH2 2.5213 6.31E−06 297 ACPL2 2.66551 1.52E−0620654 LOC648921 2.51994 1.76E−03 5325 DKK3 2.65119 5.63E−05 2445 C1ORF712.50491 9.78E−04 23853 LRRN3 2.64615 1.37E−03 21820 LOC652726 2.498515.58E−05 32615 TMSL3 2.63733 4.56E−05 16100 LOC100134868 2.492880.00076351 29822 SCG5 2.63452 1.42E−04 27105 PAPSS2 2.49026 0.00014890632000 TCF12 2.63327 1.80E−06 32246 TIMP2 2.48501 3.26E−05 3497 CCDC922.6296 2.23E−05 27648 PIP5K2B 2.48028 0.000236563 5610 DUSP1 2.608411.78E−04 12214 HSBP1 2.47723 1.82E−04 14524 LOC100130053 2.601354.53E−04 32754 TP53INP2 2.47324 2.42E−05 26279 NRSN1 2.59336 0.0011146729037 REM2 2.46751 0.000170127 25711 NANOS3 2.58916 4.15E−06 34742 ZNF842.46625 2.39E−04 5543 DPYSL4 2.58236 0.000223516 3872 CENTA1 2.461063.74E−04 6231 EXOC7 2.58163 4.89E−06 23918 LY6H 2.45793 0.00031753924134 MAPRE3 2.57689 4.85E−05 29595 RSBN1 2.45785 8.01E−04 9382HS.193784 2.57602 2.18E−06 1733 BRP44L 2.45488 5.07E−04 1608 BEX42.57251 9.83E−05 2751 C3ORF70 2.45314 1.92E−06 6514 FAM36A 2.567267.62E−05 12862 JUN 2.4481 4.37E−04 13640 LBH 2.56551 1.34E−05 15357LOC100132727 2.44481 0.00058635 9920 HS.437111 2.55902 4.44E−05 13224KIDINS220 2.43944 0.00068445 33904 WSB2 2.55865 5.08E−04 33183 TUBB4Q2.43584 0.00171503 9757 HS.369017 2.55838 4.25E−05 685 ALPP 2.432223.60E−05 18920 LOC644250 2.55607 0.000475214 14002 LOC100128274 2.430421.90E−04 27311 PDCD4 2.55148 0.000139052 20699 LOC649095 2.426550.00040167 7562 FZD1 2.54808 2.48E−06 7123 FLJ35390 2.39837 0.0016218713191 KIAA1688 2.54643 9.31E−05 16740 LOC387856 2.39594 8.21E−04 4292CNTN2 2.53802 2.29E−04 7033 FLJ22184 2.3897 4.07E−04 32474 TMEM200A2.53495 4.93E−07 20602 LOC648740 2.38616 8.38E−06 6635 FAM89B 2.53411.64E−05 10392 HS.538962 2.38583 0.000324449 23500 LOC730990 2.531973.94E−05 31615 STOX2 2.38135 4.42E−04 1375 ATP9A 2.52973 1.09E−04 23592LOC731895 2.3779 0.000838915 8126 GPR137C 2.52944 0.000217768 32863TRIM2 2.37536 5.63E−04 19248 LOC644936 2.52867 4.31E−04 30075 SERTAD42.36897 0.000363024 27855 PMP22 2.34705 7.89E−06 26135 NOL4 2.364630.000261492 27636 PINK1 2.34687 1.17E−05 27620 PIK3R1 2.35977 1.01E−073223 CAMSAP1 2.34021 1.96E−04 27073 PAK3 2.34949 1.62E−04 9323 HS.1689502.33738 1.84E−04 25716 NAP1L3 2.24879 9.43E−04 17248 LOC401115 2.333670.000151535 14359 LOC100129502 2.24469 1.52E−04 6270 F3 2.32945 2.00E−0825580 MYCBP2 2.24333 0.000445387 2256 C18ORF32 2.32914 4.41E−04 27083PAM 2.23671 0.00170991 777 ANKRD10 2.32837 2.48E−04 22583 LOC7281052.2358 0.000601193 5372 DMRTA2 2.32784 5.27E−06 29747 SARM1 2.22855.83E−07 6347 FAM117B 2.32504 9.42E−04 7602 GABRB3 2.22682 4.00E−04 731AMY1A 2.3232 0.000158787 30261 SIAH3 2.22476 1.89E−05 7610 GABRR12.31983 8.23E−08 27664 PITX1 2.22382 0.00102378 1520 BAZ2B 2.309485.18E−04 19467 LOC645452 2.21703 0.000168591 7920 GLRA2 2.30858 6.83E−056533 FAM46A 2.21591 1.24E−03 10190 HS.522924 2.30739 1.62E−03 26308NT5C2 2.21285 0.00159191 7762 GDAP1 2.30342 6.22E−05 1086 ARID5B 2.211840.000139405 31325 SPHK2 2.29983 1.68E−04 494 AFF3 2.21139 1.28E−03 31499ST18 2.2969 9.74E−04 29124 RGS16 2.21012 2.33E−05 15124 LOC1001319892.29335 1.57E−04 10056 HS.475334 2.20669 1.41E−03 23665 LOC88523 2.291710.000476299 3738 CDH10 2.20522 0.000389319 30325 SKP1 2.28675 1.76E−049819 HS.388347 2.2022 2.82E−05 2743 C3ORF58 2.28463 0.000119256 34703ZNF786 2.20193 1.65E−03 27566 PHYHIPL 2.28388 1.72E−03 8705 HIST1H4J2.19897 4.86E−06 27332 PDE4D 2.28016 3.70E−05 9740 HS.36053 2.197373.65E−05 760 ANK2 2.27803 3.84E−05 651 ALG13 2.19414 4.86E−05 1101 ARL32.26792 1.03E−03 33588 VASH2 2.19373 0.000586609 25218 MNX1 2.266314.41E−07 28810 RALGPS1 2.19311 0.000999046 7255 FLJ44048 2.264936.84E−06 5774 EFNB3 2.191 0.000919096 30029 SERINC1 2.26355 3.12E−041745 BSCL2 2.18758 8.17E−04 34800 ZSWIM6 2.2634 3.19E−04 29151 RHBDL32.18758 4.86E−05 13943 LOC100128062 2.26302 0.000498795 3740 CDH122.18674 0.000112317 18811 LOC644033 2.2611 5.93E−04 24064 MAP1LC3B2.18347 2.00E−04 20075 LOC646996 2.25787 1.36E−04 32539 TMEM59 2.177581.77E−03 7976 GNAO1 2.25676 0.00121073 33171 TUBA3E 2.17556 2.58E−0422799 LOC728661 2.25642 0.000599836 526 AGPAT4 2.17042 1.95E−04 979APPBP2 2.25324 6.41E−04 27832 PLXNA1 2.16997 0.000477137 3350 CCBE12.16311 2.69E−04 34219 ZMPSTE24 2.16821 0.00130116 5875 ELAVL1 2.158656.37E−04 34669 ZNF738 2.16494 0.00145642 13154 KIAA1370 2.15727 5.07E−0528749 RAB9B 2.16376 1.41E−03 1356 ATP6V1B2 2.15309 0.000839601 3968 CHD62.06939 4.92E−05 6457 FAM181B 2.15303 6.91E−04 34605 ZNF652 2.064410.00172675 25137 MKL2 2.14461 0.000214816 4838 CYCS 2.06436 1.39E−0327876 PNMA1 2.14384 0.000290225 34025 ZADH2 2.05857 3.02E−05 23806LRRC37B2 2.14195 4.81E−04 17246 LOC401098 2.05589 1.58E−03 6851 FGD32.139 1.84E−04 27462 PFN2 2.05533 1.58E−03 1746 BSDC1 2.138420.000430355 27137 PARP6 2.05441 5.65E−04 27997 POTEF 2.13305 0.0006550285651 DYNC1I1 2.04874 0.000834135 2969 C7ORF55 2.12929 5.66E−05 7587GABARAPL2 2.04534 0.00165234 29923 SEC11C 2.12843 8.11E−04 149 AASDHPPT2.04017 7.58E−04 31488 SSX2IP 2.12215 5.19E−06 1970 C12ORF51 2.0380.000668525 29277 RNF128 2.12203 5.41E−06 31721 SVOP 2.03603 7.00E−0430348 SLC12A2 2.12048 0.000343525 1389 ATXN1 2.03408 3.27E−05 8277 GRK52.11347 0.000106085 20667 LOC648980 2.03345 0.00062575 8573 HEPACAM22.11332 2.39E−09 24523 MIB1 2.03159 3.22E−04 1610 BEXL1 2.10951 2.62E−0431982 TCEAL3 2.03073 0.00151379 4229 CMIP 2.10246 0.000705627 12229HSD17B7 2.03003 1.50E−03 30224 SHANK2 2.10164 0.000611099 27402 PDZRN42.02983 2.33E−05 23763 LRP1B 2.09999 4.76E−05 6395 FAM13B 2.029443.18E−04 6724 FBXL16 2.09975 8.65E−04 1349 ATP6V0C 2.02572 1.08E−0326092 NLRP8 2.09701 0.00161834 12828 JAKMIP2 2.01869 8.10E−04 17496LOC440704 2.09699 0.0015232 34227 ZMYND11 2.01687 0.00171037 2543C20ORF56 2.09698 4.79E−07 31603 STMN1 2.00603 0.000940483 32624 TMX42.09279 8.41E−04 29145 RHBDD2 2.00523 6.87E−04 20006 LOC646821 2.09175.47E−05 25808 NCOA5 2.00244 0.000102715 25809 NCOA6 2.09025 1.61E−0330330 SLAIN1 2.00203 1.00E−03 2811 C5ORF28 2.08938 6.33E−04 2195C17ORF45 −2.00166 0.00119767 3279 CASD1 2.0821 6.10E−05 23469 LOC730746−2.00168 1.67E−03 28256 PRKAR1A 2.08207 0.00119569 30825 SND1 −2.004330.00128091 6208 EVI1 2.07806 4.85E−05 19456 LOC645436 −2.00559 1.69E−036920 FJX1 2.07539 1.80E−03 6242 EXOSC7 −2.00603 0.000798232 31199 SORBS22.07266 7.37E−05 17727 LOC442232 −2.00726 7.96E−05 27020 P4HA2 −2.014671.30E−03 28062 PPIH −2.00996 8.70E−04 4563 CRTAP −2.01592 2.86E−06 23234LOC729779 −2.01096 4.05E−04 29147 RHBDF1 −2.01731 9.21E−04 33239 TYK2−2.01275 0.000787726 18367 LOC643007 −2.01867 0.000867101 31614 STOX1−2.07691 0.000191887 2331 C1ORF106 −2.02039 2.06E−04 28717 RAB38−2.07726 6.33E−04 1332 ATP5G2 −2.02135 0.00169599 21994 LOC653156−2.07967 1.41E−03 25354 MRPL45 −2.02158 0.00179523 29560 RPSA −2.080960.000379606 11023 HS.552799 −2.02363 1.41E−04 17241 LOC401074 −2.081416.58E−08 17333 LOC402112 −2.02644 0.00178903 8588 HES1 −2.082850.000819562 4341 COL2A1 −2.03118 0.00112645 29494 RPLP1 −2.086881.30E−03 14518 LOC100130009 −2.03222 1.54E−05 6884 FGFR3 −2.090825.89E−06 23928 LYN −2.03236 0.000609191 4977 DBX1 −2.09957 0.00022270320489 LOC648294 −2.0327 0.0004778 29544 RPS5 −2.10042 0.00138553 32244TIMM9 −2.03469 0.0015035 29213 RIPK2 −2.10641 0.000218454 29412 RPA1−2.03493 1.70E−03 14423 LOC100129685 −2.1066 1.19E−03 33756 WDR12−2.03533 5.96E−04 27816 PLOD3 −2.10673 0.000954258 25781 NCAPD2 −2.035350.00132141 8805 HNRNPA1 −2.10967 1.74E−05 19436 LOC645385 −2.035950.00151373 6898 FHL3 −2.1127 5.26E−05 2095 C14ORF93 −2.04138 6.93E−0618250 LOC642741 −2.1147 1.02E−03 33243 TYRO3 −2.04437 4.26E−04 32523TMEM45A −2.1169 0.000464867 13396 KNTC1 −2.04704 1.95E−05 17691LOC441876 −2.11785 1.02E−03 19027 LOC644464 −2.051 0.000744833 1488BAIAP2L1 −2.11832 3.63E−05 1355 ATP6V1B1 −2.0528 0.000263628 4156 CLEC2D−2.11902 0.000733653 32263 TKT −2.05643 0.000380042 6966 FLJ10986−2.12606 0.000420546 1039 ARHGAP19 −2.05734 6.74E−05 14281 LOC100129237−2.12997 4.96E−04 4923 CYYR1 −2.05748 1.68E−05 16802 LOC388556 −2.135326.52E−04 30076 SESN1 −2.05841 2.09E−04 25385 MRPS30 −2.13565 3.28E−045054 DDX10 −2.06089 0.000505 18023 LOC642250 −2.13595 0.00107704 12782ITGB3BP −2.06492 6.73E−06 31129 SNRPA −2.13825 6.40E−04 17492 LOC440589−2.06666 4.64E−04 3730 CDCA5 −2.14343 0.0016145 23246 LOC729816 −2.068730.000791801 26121 NOB1 −2.14482 3.43E−06 24128 MAPKAPK3 −2.069330.00146997 3733 CDCA8 −2.14728 0.000158263 12221 HSD17B11 −2.071062.25E−05 33839 WDYHV1 −2.14889 0.000901318 32916 TRIM71 −2.074892.28E−05 15213 LOC100132299 −2.14934 3.42E−05 16186 LOC136143 −2.075334.81E−04 23294 LOC729964 −2.14942 0.000145725 29459 RPL27 −2.155698.13E−04 23388 LOC730246 −2.15222 5.52E−04 20894 LOC649839 −2.156370.000233819 29474 RPL36 −2.15341 1.12E−03 5649 DYM −2.15657 5.43E−0420693 LOC649076 −2.15494 0.000533848 6676 FANCG −2.15913 2.75E−04 14136LOC100128771 −2.15556 6.32E−05 26223 NPY −2.15981 4.99E−04 31588 STK24−2.23459 3.03E−04 22700 LOC728428 −2.16464 0.00122727 31024 SNORD25−2.236 5.13E−05 27540 PHGDH −2.17929 0.000926407 1282 ATIC −2.241130.000182776 23875 LSM4 −2.18409 0.00036081 33110 TTK −2.24337 4.48E−0415295 LOC100132528 −2.18468 8.34E−04 23352 LOC730107 −2.246650.000445574 14997 LOC100131609 −2.187 0.000875169 8727 HJURP −2.247692.63E−05 29442 RPL14L −2.18734 4.55E−04 23237 LOC729789 −2.248643.21E−04 16653 LOC343184 −2.19197 6.17E−05 29503 RPP40 −2.25604 1.62E−045697 EBPL −2.19209 2.05E−05 17594 LOC441246 −2.25891 4.60E−04 2763C4ORF14 −2.19304 0.00128088 17228 LOC400963 −2.26592 0.000542002 4474CPNE8 −2.19514 2.56E−04 29521 RPS18 −2.26781 0.00151457 17500 LOC440737−2.19615 0.000989795 32392 TMEM144 −2.27099 5.66E−08 22974 LOC729102−2.1963 0.000282713 7652 GALK1 −2.27206 9.50E−04 21275 LOC651149−2.19678 1.31E−03 27192 PCDH18 −2.27211 0.000113843 17403 LOC440055−2.19775 1.17E−03 4297 CNTNAP1 −2.2794 2.43E−04 29440 RPL13L −2.198172.16E−05 23041 LOC729279 −2.28142 5.88E−05 27368 PDK3 −2.20067 3.05E−045840 EIF3H −2.28165 0.000193064 31760 SYPL1 −2.20452 0.000128778 8828HNRPC −2.28204 0.00128473 14389 LOC100129585 −2.20627 8.87E−05 30493SLC27A3 −2.29001 2.85E−04 29490 RPL8 −2.20725 1.35E−04 5742 EEF1D−2.29339 0.000126152 15083 LOC100131866 −2.20753 2.67E−05 30601 SLC44A1−2.29419 0.000172089 2915 C6ORF48 −2.20788 0.000428494 3866 CENPN−2.2944 0.000143689 19976 LOC646766 −2.21365 0.00033802 29449 RPL22−2.29465 0.00105741 28801 RAI14 −2.21753 0.000675636 20804 LOC649447−2.29999 0.000262246 18557 LOC643433 −2.21779 0.000542767 28036 PPAT−2.3009 0.000393145 16862 LOC389141 −2.21829 0.000459763 25509 MTP18−2.30185 3.55E−05 5715 ECT2 −2.21838 1.41E−03 3538 CCNA2 −2.307840.000412267 29558 RPS8 −2.22068 0.00161471 33891 WNT5A −2.3104 3.36E−0419556 LOC645691 −2.22442 0.00107987 28110 PPP1R3C −2.31108 3.02E−0616693 LOC347544 −2.22526 0.000534957 1659 BMP2 −2.32278 2.31E−05 23972MAD2L1 −2.22823 0.000457464 18725 LOC643863 −2.32406 1.02E−03 18286LOC642817 −2.23024 0.00054939 359 ADA −2.3269 2.14E−05 17534 LOC440991−2.33062 1.54E−04 13751 LGMN −2.32715 4.54E−05 29559 RPS9 −2.330921.06E−04 12250 HSP90AB1 −2.32781 1.49E−03 3724 CDC7 −2.33576 0.0003429655850 EIF4B −2.32818 1.13E−03 27035 PABPC4 −2.33611 0.000164425 13724LEPREL1 −2.32851 0.000169338 33241 TYMS −2.34068 0.00106577 29457 RPL26−2.4112 0.000285349 30229 SHC1 −2.34608 0.00105777 29921 SEC11A −2.411820.00102718 21754 LOC652624 −2.34734 2.21E−04 16776 LOC388275 −2.412885.56E−06 5846 EIF3M −2.34987 0.00181367 29438 RPL13A −2.415920.000922424 15032 LOC100131713 −2.35114 0.000201749 3795 CDKN3 −2.418560.000112421 29432 RPL10A −2.35345 0.00131867 30404 SLC20A2 −2.418796.23E−06 24235 MCM6 −2.35636 0.00108431 32648 TNFRSF19 −2.42065 6.93E−0519046 LOC644511 −2.36244 0.000514797 31239 SP5 −2.42085 2.37E−09 5044DDIT4L −2.36718 0.000107365 23974 MAD2L2 −2.42585 0.000123115 545 AHCY−2.3677 0.000431822 12602 IMPA2 −2.42996 1.90E−06 18592 LOC643531−2.37199 1.01E−03 17525 LOC440927 −2.43391 0.00119949 5744 EEF1G−2.37611 0.000630065 29414 RPA3 −2.43719 0.00177478 5765 EFHD1 −2.380431.20E−03 16156 LOC127295 −2.43999 5.06E−04 32637 TNFRSF10B −2.380661.53E−05 29565 RPUSD3 −2.44425 1.98E−07 26099 NME1-NME2 −2.384264.44E−05 16979 LOC390578 −2.44648 8.18E−08 22757 LOC728564 −2.384943.69E−04 15104 LOC100131940 −2.44655 2.19E−05 8826 HNRPA1P4 −2.386093.87E−05 26103 NME4 −2.44757 0.00114224 17458 LOC440359 −2.386915.63E−04 8825 HNRPA1L-2 −2.45235 5.46E−04 28560 PTPN13 −2.392110.000413752 1537 BCAR3 −2.45546 0.000368573 15180 LOC100132199 −2.393372.68E−05 14206 LOC100129028 −2.45901 3.51E−04 4955 DARS −2.397281.56E−03 16672 LOC345041 −2.46013 0.000101781 1882 CORF1 −2.398282.51E−05 3125 CA14 −2.46417 0.000205741 23282 LOC729926 −2.399737.65E−04 32089 TET1 −2.46774 3.21E−05 15041 LOC100131735 −2.400352.35E−05 12231 HSD17B8 −2.47022 0.000519001 22829 LOC728732 −2.400896.11E−05 4479 CPS1 −2.47967 2.21E−06 3973 CHEK1 −2.40178 1.16E−06 25464MTA3 −2.48169 8.09E−05 600 AKR1A1 −2.40346 0.000695089 5758 EFEMP1−2.48833 1.42E−05 15586 LOC100133372 −2.40481 1.53E−04 28160 PQLC3−2.48845 0.000585543 31607 STOM −2.40533 3.40E−07 30245 SHMT2 −2.489640.00117427 22710 LOC728453 −2.40686 0.0001068 23103 LOC729423 −2.500416.49E−06 23391 LOC730255 −2.50972 1.22E−03 12741 ISG20L1 −2.504049.70E−05 24097 MAP4K2 −2.51767 5.17E−06 29539 RPS3 −2.50954 0.0001034467686 GAPDH −2.51826 0.00128279 22857 LOC728791 −2.5096 5.69E−06 2433C1ORF57 −2.51861 1.49E−03 15975 LOC100134393 −2.6327 2.71E−04 2262C18ORF56 −2.51885 0.000156088 9745 HS.363526 −2.6341 0.000643774 1792BUB1 −2.52119 9.50E−06 561 AIF1L −2.63663 1.37E−04 5831 EIF2S3 −2.523733.38E−04 8597 HEXB −2.63709 1.51E−03 29039 RENBP −2.53102 4.92E−07 5993EPDR1 −2.63912 0.00156376 29481 RPL39L −2.53153 0.00013954 7966 GMPS−2.64155 1.99E−04 17141 LOC399804 −2.5389 3.38E−05 27563 PHYH −2.643222.14E−05 34552 ZNF581 −2.54051 9.37E−05 4100 CKS1B −2.64512 1.68E−041156 ARRDC4 −2.54054 2.88E−05 7898 GLDC −2.64809 0.00143371 8789 HMMR−2.54277 8.17E−05 5513 DPH5 −2.6481 2.11E−05 28953 RBMX −2.5493 5.57E−0518914 LOC644237 −2.66097 0.0011589 32179 THEM2 −2.55298 5.30E−04 20185LOC647285 −2.66563 1.82E−04 13239 KIF20A −2.55316 1.13E−05 22536LOC727984 −2.67684 0.000223687 1403 AURKA −2.55681 5.93E−05 8625 HIBADH−2.68374 0.00144114 27066 PAICS −2.55763 0.00111653 684 ALPL −2.692662.39E−06 5836 EIF3D −2.56061 6.72E−05 17636 LOC441506 −2.70546 6.10E−0513252 KIF2C −2.56757 1.70E−06 30602 SLC44A2 −2.70757 1.24E−03 14814LOC100130980 −2.57639 4.33E−05 16741 LOC387867 −2.71424 2.45E−05 31821TAF1D −2.57677 6.27E−06 23060 LOC729340 −2.7157 0.000999351 27653 PIR−2.58392 2.26E−06 27743 PLCD1 −2.72 1.14E−07 7564 FZD2 −2.585180.000146094 22597 LOC728139 −2.72434 4.77E−05 28778 RAD51AP1 −2.591045.06E−06 28530 PTGR1 −2.72675 7.39E−05 14695 LOC100130562 −2.595633.41E−05 5741 EEF1B2 −2.7289 4.84E−05 8547 HEATR1 −2.59617 5.95E−0718360 LOC642989 −2.73236 0.000921883 22591 LOC728126 −2.59998 3.37E−0520058 LOC646942 −2.74335 5.41E−05 29483 RPL4 −2.62451 7.56E−05 16737LOC387825 −2.74954 5.93E−06 25783 NCAPG −2.62632 6.41E−05 16236LOC148430 −2.76485 2.32E−05 20366 LOC647856 −2.6267 1.31E−05 29441 RPL14−2.76505 4.49E−06 29462 RPL29 −2.62809 4.36E−07 31176 SNX5 −2.774264.41E−08 23274 LOC729903 −2.63121 4.28E−05 13592 LAMA1 −2.77535 2.89E−0727370 PDLIM1 −2.79852 0.000250318 16814 LOC388707 −2.77694 1.99E−0627030 PABPC1 −2.7998 0.000107149 29179 RHPN2 −2.78226 0.00110049 7486FRZB −2.81189 5.35E−04 23133 LOC729500 −2.78295 8.63E−06 17023 LOC391075−2.82637 0.00139394 2524 C20ORF199 −2.79683 8.26E−05 6705 FBL −2.82762.59E−05 23373 LOC730187 −3.08276 4.32E−06 13708 LEF1 −2.83207 1.15E−0525214 MND1 −3.08698 3.39E−07 4101 CKS2 −2.83376 0.000537912 14412LOC100129657 −3.08986 0.00120608 13438 KRT19 −2.83803 2.34E−05 33282UBE2C −3.09218 4.69E−06 28410 PSAT1 −2.8451 8.29E−07 30114 SFRP1−3.10477 0.000250519 33979 YAP1 −2.84569 0.00106156 17015 LOC391019−3.10602 2.05E−05 29435 RPL12 −2.85091 4.23E−05 28602 PTTG1 −3.113092.05E−05 22553 LOC728031 −2.85482 8.14E−05 17541 LOC441013 −3.118666.73E−07 29524 RPS2 −2.85918 1.32E−05 20657 LOC648931 −3.12515 3.24E−0923861 LRTM1 −2.85972 1.48E−12 31661 SUCLG2 −3.13505 5.75E−07 26404NUSAP1 −2.86459 9.00E−05 1559 BCL2L12 −3.13599 4.40E−07 20473 LOC648249−2.86802 4.01E−06 32222 TIGA1 −3.13783 7.09E−05 23319 LOC730029 −2.876073.58E−05 7770 GDF15 −3.14204 1.42E−07 24516 MGST1 −2.87812 2.82E−06 2878C6ORF160 −3.15022 0.000111744 349 ACVR1 −2.88477 0.000447642 15382LOC100132795 −3.21797 1.51E−05 30500 SLC29A1 −2.91375 2.94E−06 4930 DAB2−3.2203 2.91E−05 17086 LOC391833 −2.91531 4.06E−05 32933 TRIP6 −3.231321.32E−06 20614 LOC648771 −2.91567 7.23E−05 20091 LOC647030 −3.237314.86E−05 27493 PGM1 −2.92082 0.000287518 26048 NKD1 −3.24181 4.37E−0630836 SNHG6 −2.92725 2.13E−05 6320 FAM107A −3.24905 0.000449795 20015LOC646849 −2.95184 3.75E−05 16552 LOC286444 −3.25137 6.05E−06 16783LOC388339 −2.95309 4.55E−05 26390 NUP37 −3.26236 3.70E−05 3785 CDKN1A−2.95807 0.000310093 19783 LOC646294 −3.28211 1.06E−06 29436 RPL12P6−2.97114 2.40E−06 2210 C17ORF61 −3.28907 2.68E−05 5258 DIMT1L −2.978031.83E−06 19553 LOC645688 −3.29172 3.95E−06 15940 LOC100134304 −2.982234.08E−08 25847 NCRNA00219 −3.29327 7.23E−05 13733 LGALS1 −2.984730.00160731 28663 QPRT −3.29832 2.04E−05 27902 PODXL −3.00627 2.35E−0831361 SPON1 −3.32269 5.27E−06 16630 LOC341315 −3.02065 1.10E−04 30835SNHG5 −3.32459 0.000204237 481 ADSS −3.02244 4.06E−06 4596 CSDA −3.36290.000363294 29557 RPS7 −3.06985 2.69E−06 455 ADM −3.40482 4.11E−05 1406AURKB −3.07612 5.67E−07 23228 LOC729768 −3.41997 2.20E−05 6707 FBLN1−3.53279 3.88E−06 1753 BST2 −3.42742 1.17E−07 28241 PRIM1 −3.538921.17E−06 20685 LOC649049 −3.46913 1.63E−06 8754 HLA-E −3.54293 2.03E−0717292 LOC401537 −3.50501 3.79E−07 643 ALDOA −3.55096 0.000184641 19348LOC645173 −4.02454 2.71E−06 14422 LOC100129681 −3.55393 9.42E−07 28605PTTG3P −4.02805 3.37E−06 8773 HMGB2 −3.56419 9.08E−05 4239 CMTM7−4.08386 7.09E−09 32964 TRPM4 −3.5945 2.41E−06 8089 GPI −4.088211.18E−05 12398 IFITM2 −3.59918 1.38E−03 1616 BGN −4.11601 0.00034809532177 THBS4 −3.6116 6.59E−07 23196 LOC729679 −4.2525 4.55E−08 3540CCNB1IP1 −3.62989 3.35E−08 3694 CDC20 −4.28351 3.38E−06 5477 DOCK10−3.64495 7.09E−07 32729 TOP2A −4.29945 1.43E−06 28387 PRSS23 −3.672530.000258012 30828 SNHG1 −4.38641 2.77E−06 26369 NUDT7 −3.67565 7.62E−053243 CAPN6 −4.44794 4.50E−07 26207 NPM3 −3.69114 1.35E−06 30284 SILV−4.59486 1.80E−07 23887 LTA4H −3.69913 6.82E−05 15342 LOC100132673−4.69254 2.18E−07 12399 IFITM3 −3.70541 0.000779898 29716 SALL4 −4.959092.66E−08 8769 HMGA1 −3.72752 3.30E−08 3541 CCNB2 −5.23718 4.33E−08 323ACTA2 −3.74071 1.75E−04 30511 SLC2A3 −5.39887 5.64E−07 1530 BBS9 −3.76363.82E−07 13691 LDHA −5.76362 0.000569306 7501 FSTL1 −3.80754 0.0001369617500 FST −5.82949 8.74E−09 27805 PLIN2 −3.82123 4.77E−10 1543 BCAT1−6.13131 3.52E−06 28975 RBPMS2 −3.82727 3.95E−06 5323 DKK1 −6.174934.94E−08 23218 LOC729742 −3.84552 7.82E−07 4343 COL4A1 −6.21986 6.18E−0713597 LAMB1 −3.85107 1.24E−07 8078 GPC3 −6.35513 0.000466041 6291 FABP7−3.85422 0.000335025 4333 COL22A1 −6.371 1.87E−08 12606 IMPDH2 −3.859524.79E−06 32774 TPM2 −6.8139 1.85E−08 13834 LITAF −3.86 4.66E−05 2430C1ORF54 −7.81915 1.70E−07 3953 CHCHD10 −3.91295 4.20E−06 965 APOE−9.10671 6.03E−07 2439 C1ORF64 −3.9544 5.48E−10 13804 L1N28 −26.88211.20E−13 8473 HAUS4 −3.96227 4.95E−12 925 APCDD1 −3.97854 6.09E−08 5952ENO3 −4.00663 6.90E−09 27019 P4HA1 −4.01147 0.00032695 4724 CTSL2−4.01208 1.47E−06

TABLE 4 Gene expression array data of significantly up-regulated anddown- regulated genes at differentiation day 25 in SHH/FGF8/Chir treatedFloor-plate based population over control LSB treated population. ColumnColumn Fold- Column Column Fold- # ID Change p-value # ID Change p-value32126 TFF3 25.0585 2.47E−14 26928 OSBPL10 3.27941 3.81E−07 1209 ASCL114.9328 1.97E−07 5372 DMRTA2 3.26437 1.81E−08 7395 FOXA1 12.98974.15E−14 33930 XBP1 3.2414 0.000921089 29133 RGS4 11.0181 6.65E−09 27664PITX1 3.23009 1.10E−05 27988 POSTN 8.20403 2.16E−10 9819 HS.3883473.14523 8.26E−08 7396 FOXA2 7.98132 1.60E−14 30239 SHISA2 3.134114.23E−05 4342 COL3A1 7.58625 2.49E−05 2743 C3ORF58 3.10109 1.50E−0612731 IRX3 7.42399 1.64E−06 623 ALCAM 3.00487 0.000307411 9374 HS.191937.27196 7.36E−09 6533 FAM46A 2.99243 3.45E−05 3978 CHGA 7.14305 2.77E−0828590 PTPRO 2.94164 4.45E−05 2470 C1QL1 7.1366 1.38E−09 18750 LOC6439112.93448 1.14E−06 30390 SLC18A1 7.04751 3.52E−14 31669 SULF2 2.923978.02E−05 22823 LOC728715 6.5801 8.79E−09 5348 DLL1 2.92274 8.36E−0512456 IGFBP5 6.15962 3.65E−05 30066 SERPINF1 2.76873 0.000156193 27665PITX2 6.10679 6.79E−14 5349 DLL3 2.75425 0.000173675 12655 INSM2 5.184381.70E−09 3186 CALCA 2.71125 2.10E−07 12944 KCNJ16 4.98753 8.89E−13 30590SLC39A8 2.67444 2.25E−05 436 ADCYAP1 4.94597 2.43E−09 32000 TCF122.66777 1.46E−06 31361 SPON1 4.93795 4.69E−08 3941 CGNL1 2.638480.00120585 13857 LMO3 4.83578 7.25E−08 32769 TPH1 2.62647 1.95E−06 13865LMX1A 4.63407 1.51E−06 25941 NENF 2.55992 0.000599552 2543 C20ORF564.4431 3.63E−13 29509 RPRM 2.53138 0.00135525 12733 IRX5 4.2929 1.45E−0632144 TGFBR3 2.46754 8.27E−07 7988 GNB3 4.13634 1.52E−10 13689 LDB22.40983 2.56E−05 32159 TH 4.08743 0.000370318 2850 C6ORF117 2.40090.000508473 5346 DLK1 4.02203 1.73E−05 31806 TACSTD1 2.38661 0.0015449531775 SYT4 4.00669 0.000372793 24375 MFNG 2.31488 5.04E−05 9428HS.204481 3.88697 6.71E−09 5424 DNAJC19 2.30405 4.42E−07 6197 ETS23.70155 3.07E−09 12986 KCNS3 2.28802 0.000136062 5036 DDC 3.612540.000194062 27855 PMP22 2.27371 1.36E−05 26250 NR4A2 3.55879 9.29E−104003 CHN2 2.26467 0.000550787 2867 C6ORF141 3.48146 6.97E−09 5691 EBF32.26453 0.000571924 2245 C18ORF10 −2.00297 0.00129421 3187 CALCB 2.113592.49E−06 28546 PTN −2.003 0.000354472 28325 PROX1 2.08192 0.0001358725920 EMID2 −2.00456 1.77E−05 25218 MNX1 2.05528 3.37E−06 24405 MGC11082−2.00498 2.75E−05 6270 F3 2.01261 5.71E−07 5949 ENKUR −2.007420.000107398 12971 KCNMB4 −2.09934 0.00140267 26398 NUP93 −2.011110.00124009 3125 CA14 −2.10277 0.00147559 10837 HS.545615 −2.013782.65E−05 17153 LOC399959 −2.10829 0.000816932 12688 IQCC −2.013830.00019749 15855 LOC100134073 −2.10866 0.000366572 6196 ETS1 −2.017510.000848186 26322 NTM −2.10973 0.000159062 27290 PCSK5 −2.020790.00122771 7595 GABRA2 −2.11173 0.00169771 16112 LOC100192378 −2.02164.35E−05 5618 DUSP18 −2.11289 8.90E−05 19806 LOC646345 −2.023110.000183202 4289 CNTFR −2.11375 0.00132713 23846 LRRIQ1 −2.027180.000541683 3760 CDH8 −2.11381 7.41E−05 27746 PLCE1 −2.02761 0.0002172813196 CALM1 −2.11418 0.00157715 2727 C3ORF39 −2.03023 0.000131521 13615LARGE −2.11685 2.37E−05 26072 NLGN3 −2.03154 2.45E−05 32933 TRIP6−2.11829 0.00047459 2770 C4ORF22 −2.03489 0.00113778 2242 C17ORF97−2.12666 0.000851745 3491 CCDC88C −2.03515 0.00112864 5478 DOCK11−2.12763 0.000306132 25751 NAV3 −2.03716 8.63E−05 7377 FNDC4 −2.133380.000482465 30413 SLC22A17 −2.03749 0.000703407 29672 RYR3 −2.135730.000392208 13014 KDELC2 −2.04133 0.00147391 30669 SLC7A6 −2.138650.000299041 27749 PLCH1 −2.0426 0.000234672 29816 SCD5 −2.14266 9.40E−0516489 LOC284988 −2.04353 0.000785305 12212 HS6ST2 −2.14751 0.000204739938 HS.440518 −2.04573 0.000285223 4213 CLSTN2 −2.15117 2.14E−05 28076PPM1H −2.04637 0.00038276 16924 LOC389816 −2.15626 4.60E−09 429 ADCY3−2.05728 0.000712376 2102 C15ORF26 −2.15663 0.000242005 31199 SORBS2−2.05812 8.31E−05 1103 ARL4C −2.16184 0.00049625 17624 LOC441453 −2.06763.05E−06 31151 SNX10 −2.16614 0.000623725 13793 LIM2 −2.07092 0.0004064116382 LOC255783 −2.17123 0.000592438 33165 TUB −2.07384 0.00018715630372 SLC16A14 −2.17701 0.000382805 3462 CCDC65 −2.07613 0.0015151613177 KIAA1598 −2.1779 0.000634174 4305 COBL −2.07819 0.000485834 28587PTPRM −2.17848 0.000334455 6912 FILIP1 −2.07924 9.69E−05 8661 HIST1H2AC−2.18157 0.00018782 5195 DGCR6 −2.07943 0.000935686 30015 SEPT6 −2.184830.00101243 33905 WSCD1 −2.08212 0.00033792 6572 FAM65B −2.18593 1.46E−0512933 KCNIP1 −2.08721 3.72E−05 4614 CSMD2 −2.18777 0.000579739 4933DACH2 −2.09051 9.24E−05 19737 LOC646168 −2.18864 0.000417767 25960NEUROG2 −2.09361 0.00111856 2429 C1ORF53 −2.19182 0.0017844 25930 NEK2−2.09683 0.00144313 17855 LOC641785 −2.19315 0.000255891 3288 CASP3−2.09925 0.000130992 5570 DSCR6 −2.20122 0.00137487 30388 SLC17A8−2.21639 0.000916084 13835 LIX1 −2.20141 6.32E−06 26228 NQO1 −2.219740.000753734 23979 MAF −2.20457 6.38E−05 23817 LRRC46 −2.221590.000756129 4734 CTXN1 −2.21039 0.0007813 32380 TMEM132D −2.226620.000463067 2049 C14ORF159 −2.2127 0.000171696 2705 C3ORF15 −2.22990.000615847 28906 RBKS −2.35486 1.25E−05 27781 PLEKHG1 −2.23885 1.49E−053806 CDRT4 −2.36622 0.000173226 29607 RSPH9 −2.24091 0.000109859 32129TFPI −2.36876 0.000644776 23965 MAB21L1 −2.24222 2.00E−05 5763 EFHC1−2.36977 0.00112503 25942 NEO1 −2.24511 2.27E−05 3746 CDH18 −2.378216.60E−06 34157 ZFP106 −2.24688 0.00129693 33006 TSHZ3 −2.384150.00160322 33083 TTC29 −2.24927 0.000317648 6901 FHOD3 −2.385720.00106294 3415 CCDC19 −2.25412 0.000638581 13805 LIN28B −2.386880.000393414 30209 SH3GL2 −2.2573 0.00175354 1806 C10ORF107 −2.397350.000750154 839 ANKS1B −2.25784 6.46E−05 32034 TCTEX1D1 −2.409880.00160003 3472 CCDC74A −2.25911 3.66E−05 31146 SNTN −2.428930.000945171 27561 PHTF1 −2.26039 0.00113655 30057 SERPINB6 −2.433051.01E−05 26505 OPCML −2.26062 2.34E−05 28580 PTPRD −2.43513 6.24E−0523859 LRRTM4 −2.26387 0.00050229 28210 PRDM8 −2.44594 0.000113613 27040PACRG −2.26701 8.08E−05 2614 C22ORF15 −2.45467 0.000112958 24318 MELK−2.27019 0.000423662 12892 KBTBD9 −2.45472 2.16E−06 10017 HS.452398−2.27201 0.000477927 6246 EXT1 −2.4653 0.000117948 9237 HS.147562−2.27279 0.000798954 25946 NETO2 −2.46601 0.000568595 26976 OTX1−2.27327 0.000877908 493 AFF2 −2.4707 0.0012124 8046 GOLSYN −2.277240.000100581 31610 STOML3 −2.48765 0.000560404 1074 ARHGEF6 −2.279820.000748746 31577 ST1L −2.49103 0.000199457 4639 CSRNP3 −2.283610.000213299 4826 CYB5D1 −2.49147 0.000128822 27547 PHLDA1 −2.286672.70E−05 2078 C14ORF45 −2.49501 0.000787004 3537 CCNA1 −2.28864 6.92E−0533501 USP13 −2.49771 6.06E−05 26071 NLGN2 −2.29131 0.000215422 34226ZMYND10 −2.50015 0.000105158 5325 DKK3 −2.29195 0.000372444 28507 PTCHD1−2.50016 0.00120417 6326 FAM108C1 −2.29624 0.000567659 5322DKFZP781N1041 −2.50483 0.000661286 28721 RAB3B −2.30577 0.000211905 3253CAPSL −2.50626 0.000844682 2372 C1ORF158 −2.3174 0.00184147 14289LOC100129268 −2.508 0.000176359 32499 TMEM231 −2.32173 0.000378145 17551LOC441054 −2.5088 0.00166136 5550 DRD1IP −2.3265 0.00129147 26395NUP62CL −2.51081 0.000373281 12785 ITGB5 −2.32837 0.00143999 1776 BTG3−2.51582 0.00131261 30937 SNORA79 −2.32935 0.000394221 33005 TSHZ2−2.51649 3.77E−05 2538 C20ORF46 −2.34101 0.000827542 4705 CTNND2−2.52798 0.00038507 5939 EMX2OS −2.34991 1.28E−05 29792 SCARNA11−2.53148 3.35E−06 3153 CACNA1E −2.35127 0.000755994 4965 DBC1 −2.532210.000370596 728 AMPH −2.55801 6.39E−06 32507 TMEM31 −2.53343 2.39E−0633718 VWC2 −2.56363 0.000340976 1927 C11ORF75 −2.53649 0.000498243 13724LEPREL1 −2.56798 4.43E−05 6740 FBXO15 −2.54184 0.00139562 1057 ARHGDIB−2.573 5.20E−06 27370 PDLIM1 −2.54956 0.000681773 5765 EFHD1 −2.592940.000474204 25939 NELL1 −2.7509 0.00086776 3758 CDH6 −2.6075 0.00030326832531 TMEM51 −2.77628 6.84E−06 7043 FLJ23152 −2.60944 0.000143522 15778LOC100133887 −2.78135 1.17E−05 31282 SPATA17 −2.61656 0.00020388 815ANKRD38 −2.79552 0.000168264 7106 FLJ33590 −2.61873 0.00025779 33758WDR16 −2.80194 0.000532777 16264 LOC151162 −2.62005 0.000666466 12801ITM2C −2.81062 7.32E−05 32440 TMEM178 −2.62313 0.00011953 32847 TRH−2.84178 0.00148109 1987 C12ORF69 −2.62861 5.06E−06 2881 C6ORF165−2.84456 6.28E−05 2970 C7ORF57 −2.63494 0.000118643 15644 LOC100133542−2.84572 9.65E−05 23830 LRRC6 −2.63632 0.000197619 29811 SCARNA8−2.84646 9.99E−06 6173 ESM1 −2.63913 0.000368518 33964 XPR1 −2.853525.49E−06 153 ABAT −2.64114 3.81E−06 25940 NELL2 −2.86423 0.00024831 2793C4ORF47 −2.64571 0.00134045 5455 DNER −2.87832 0.00127284 2301 C19ORF51−2.64617 7.44E−05 33807 WDR63 −2.88707 4.43E−05 34155 ZFHX4 −2.661275.12E−06 3038 C9ORF116 −2.89396 0.000215965 5762 EFHB −2.663380.000148296 2553 C20ORF85 −2.89405 7.84E−05 6920 FJX1 −2.66433 8.09E−055531 DPY19L1 −2.90287 0.000183171 12872 KANK4 −2.66744 0.000457332 31955TBR1 −2.90815 7.60E−06 26104 NME5 −2.66836 0.000118819 8655 HIST1H1C−2.91043 9.46E−05 3148 CACHD1 −2.67846 0.000100474 3401 CCDC146 −2.910570.000155147 25628 MYO16 −2.67972 8.67E−05 31935 TBC1D9 −2.91662 6.93E−0528838 RAPGEF2 −2.68049 7.06E−05 7421 FOXJ1 −2.94694 0.000577632 5553DRD4 −2.68408 0.000500735 32783 TPPP3 −2.95648 0.000353912 4087 CITED2−2.68818 0.00192224 5337 DLG2 −2.96987 1.13E−06 23790 LRRC26 −2.688892.44E−06 27810 PLK4 −2.98102 1.71E−06 2631 C22ORF42 −2.69089 0.0003938224315 MEIS2 −3.03243 0.00109977 25564 MXRA5 −2.69302 0.00192861 31243SPA17 −3.03709 0.000523067 12836 JAZF1 −2.70808 5.58E−05 10979 HS.551307−3.04712 1.13E−05 13346 KLHL29 −2.71506 5.11E−06 29794 SCARNA13 −3.055684.35E−05 25749 NAV1 −2.71696 0.000248061 32378 TMEM132B −3.064081.54E−06 29306 RNF175 −2.71799 3.04E−05 2690 C2ORF77 −3.07752 4.19E−0513815 LINGO2 −2.71868 2.30E−05 27660 PITPNM1 −3.09871 2.62E−05 25208MMRN1 −2.73481 0.000116097 32963 TRPM3 −3.10437 0.000937085 3918 CFDP1−2.73575 0.000150699 3640 CD36 −3.10624 0.000121554 31242 SP8 −2.738625.04E−05 26070 NLGN1 −3.1408 9.93E−05 1311 ATP1B3 −2.7498 8.64E−05 5749EFCAB1 −3.15705 0.000423897 5681 E2F7 −3.17981 2.36E−07 3121 CA10−3.15947 0.000537722 8174 GPR37 −3.18311 2.85E−05 3215 CAMK2N1 −3.161810.000561888 29510 RPRML −3.19177 0.000271322 8016 GNRH1 −3.165541.25E−05 4349 COL4A6 −3.1938 5.88E−05 13037 KHDRBS3 −3.16572 0.00026943727887 PNOC −3.19863 4.88E−05 32270 TLE4 −3.57637 7.16E−05 1543 BCAT1−3.20615 0.000858798 28981 RCAN2 −3.61105 0.000608261 28813 RALYL−3.21662 0.000509498 32069 TEKT1 −3.61846 8.36E−05 30531 SLC32A1−3.24737 0.0002618 17491 LOC440585 −3.62588 0.000175217 25216 MNS1−3.25707 0.000109366 5660 DYNLRB2 −3.6297 6.28E−05 3364 CCDC109B−3.26787 0.000147649 5938 EMX2 −3.66175 1.17E−06 4004 CHODL −3.289620.00114785 24314 MEIS1 −3.68295 0.000402893 965 APOE −3.29237 0.0015557813856 LMO2 −3.74164 7.37E−06 5764 EFHC2 −3.29611 0.000143319 6004 EPHB1−3.77917 0.000119962 29002 RDH10 −3.30034 0.000369857 7504 FSTL5−3.80048 0.000392914 29600 RSHL3 −3.30753 6.17E−05 1119 ARMC3 −3.821422.66E−05 12317 HTRA1 −3.31522 0.000437969 31257 SPAG6 −3.86214 1.74E−052398 C1ORF194 −3.31829 1.21E−05 7865 GJA1 −3.88524 0.000511853 5895ELMOD1 −3.32218 0.000142072 3274 CASC1 −3.88963 2.00E−05 3066 C9ORF171−3.33251 4.78E−05 7696 GAS1 −3.95396 2.45E−07 33592 VAT1L −3.34020.000247714 7805 GFRA2 −4.00209 0.000116201 12399 IFITM3 −3.35420.0016376 3050 C9ORF135 −4.09611 3.21E−05 28319 PROM1 −3.35504 5.84E−0523853 LRRN3 −4.2728 1.34E−05 29034 RELN −3.43956 1.30E−05 29603 RSPH1−4.35627 1.08E−05 2397 C1ORF192 −3.45738 0.000124675 6458 FAM183A−4.40302 2.73E−05 586 AKAP14 −3.46559 1.94E−05 3220 CAMKV −4.475434.97E−05 5032 DDAH1 −3.46896 1.42E−05 27284 PCP4 −4.64191 1.37E−05 3766CDK2AP2 −3.4706 3.31E−06 29723 SAMD3 −4.7132 2.41E−06 3652 CD47 −3.473346.18E−05 31223 S0X3 −4.74012 8.45E−06 4283 CNR1 −3.48521 9.70E−05 29396ROPN1L −4.86578 3.17E−06 12694 IQCG −3.4934 2.59E−05 29972 SEMA3C−5.03965 4.16E−05 10300 HS.537002 −3.49808 0.000142247 3695 CDC20B−5.17405 5.72E−07 12506 IL13RA2 −3.50471 5.79E−05 4298 CNTNAP2 −5.339024.01E−06 28709 RAB31 −3.51372 7.79E−05 2006 C13ORF30 −5.4414 1.12E−0628536 PTH2 −3.54413 0.000188892 33600 VCAN −5.47499 0.000917643 31283SPATA18 −3.54742 5.71E−05 1936 C11ORF88 −5.55748 2.96E−06 3071 C9ORF24−3.55395 9.04E−05 30115 SFRP2 −6.27713 3.81E−08 7432 FOXN4 −3.572995.14E−09 27159 PAX6 −6.64236 3.94E−10 27171 PBX3 −3.57487 0.000447274866 ANXA1 −7.3691 4.96E−06 3561 CCNO −9.3158 2.19E−08 13768 LHX2−9.53566 3.56E−05

TABLE 5 Gene expression array data of significantly up-regulated anddown- regulated genes at differentiation day 25 in SHH/FGF8/Chir treatedFloor- plate based population over SHH/FGF8 only treated population.Column Column Fold- p-value Column Column Fold- # ID Change # ID Changep-value 32126 TFF3 24.0181 3.37E−14 32000 TCF12 5.05883 1.27E−10 1209ASCL1 22.1637 1.69E−08 4003 CHN2 4.53859 9.76E−08 9374 HS.19193 14.70451.42E−11 2867 C6ORF141 4.43629 1.97E−10 7395 FOXA1 13.8748 2.30E−1432417 TMEM163 4.40645 3.65E−08 13865 LMX1A 10.953 4.33E−10 26245 NR2F24.38488 8.32E−05 12456 IGFBP5 9.56964 1.58E−06 5036 DDC 4.37185 3.55E−054342 COL3A1 9.49528 5.64E−06 6700 FAT3 4.28732 4.74E−06 12361 ID39.45077 3.33E−08 9428 HS.204481 4.25031 1.93E−09 27988 POSTN 9.104537.82E−11 8288 GRM8 4.16549 1.67E−06 7396 FOXA2 8.85174 5.07E−15 28590PTPRO 4.13365 8.06E−07 3941 CGNL1 8.71553 1.37E−08 32159 TH 4.108760.000355952 30390 SLC18A1 8.37911 4.77E−15 31361 SPON1 4.063 4.56E−075689 EBF1 8.02385 2.37E−06 34166 ZFP36L1 4.05459 0.000219198 2470 C1QL17.76325 5.90E−10 13689 LDB2 3.9497 1.90E−08 22823 LOC728715 7.732461.77E−09 32057 TEAD2 3.94608 6.68E−06 27665 PITX2 7.32855 7.16E−15 12731IRX3 3.93674 0.000270763 12655 INSM2 7.04572 4.97E−11 28942 RBM473.91109 0.000154758 26250 NR4A2 6.75415 1.28E−13 30066 SERPINF1 3.842763.72E−06 8156 GPR177 6.28979 1.08E−05 1666 BMP7 3.83782 1.66E−05 13857LMO3 6.20624 4.64E−09 25444 MSX1 3.74709 3.45E−05 33930 XBP1 5.84366.74E−06 8593 HES6 3.66092 5.43E−05 12944 KCNJ16 5.71955 1.37E−13 12149HS.7023 3.65165 0.000170814 27821 PLS3 5.62251 8.45E−07 30239 SHISA23.60636 8.64E−06 5372 DMRTA2 5.61111 8.09E−12 9819 HS.388347 3.523161.46E−08 7988 GNB3 5.55425 2.56E−12 7564 FZD2 3.48734 3.69E−06 31669SULF2 5.51821 6.78E−08 26928 OSBPL10 3.44485 1.91E−07 33128 TTR 5.482342.88E−09 27664 PITX1 3.40016 5.92E−06 2543 C20ORF56 5.42131 2.00E−142743 C3ORF58 3.31658 5.91E−07 32762 TPBG 5.30583 7.23E−05 30590 SLC39A83.30002 1.28E−06 436 ADCYAP1 5.20834 1.29E−09 32144 TGFBR3 3.287276.00E−09 26977 OTX2 5.10526 6.89E−06 18750 LOC643911 3.2583 2.43E−073978 CHGA 5.07311 7.78E−07 17491 LOC440585 3.24609 0.000469294 27772PLEKHA5 3.09727 0.000150905 24375 MFNG 3.24122 3.31E−07 26335 NUAK13.08699 1.10E−05 5346 DLK1 3.21349 0.000161289 34155 ZFHX4 3.054636.49E−07 7562 FZD1 3.15302 8.07E−08 27855 PMP22 3.03785 1.12E−07 3186CALCA 3.14299 1.75E−08 4491 CPVL 3.02972 0.000317992 7853 GINS2 2.35417.75E−05 7421 FOXJ1 3.01784 0.000461499 29887 SDC2 2.34674 3.00E−05 2850C6ORF117 3.00725 3.47E−05 27402 PDZRN4 2.29374 2.31E−06 27105 PAPSS22.97981 1.49E−05 16112 LOC100192378 2.2867 4.57E−06 12358 ID1 2.943610.000160308 21088 LOC650494 2.27924 4.94E−06 33004 TSHZ1 2.936372.29E−07 8583 HERC5 2.27663 2.89E−07 5691 EBF3 2.92465 2.26E−05 1775BTG2 2.27014 6.72E−06 1086 ARID5B 2.92012 2.33E−06 30719 SLK 2.249973.57E−05 32769 TPH1 2.88914 4.24E−07 19806 LOC646345 2.232 3.69E−05 5424DNAJC19 2.87234 6.43E−09 25137 MKL2 2.22229 0.00012672 6208 EVI1 2.860692.04E−07 7610 GABRR1 2.21011 2.33E−07 13858 LMO4 2.85216 4.81E−05 8573HEPACAM2 2.17226 1.17E−09 12733 IRX5 2.84963 0.000139911 29151 RHBDL32.16408 5.80E−05 5879 ELF1 2.78573 1.43E−06 27016 P2RY5 2.16065 5.91E−0623752 LRIG1 2.77491 0.000210665 30261 SIAH3 2.14795 3.46E−05 23822LRRC4C 2.77041 5.37E−07 30517 SLC2A8 2.13975 8.81E−05 4780 CXCR4 2.724685.12E−07 7457 FREM1 2.12587 7.87E−08 27696 PKNOX2 2.7166 1.59E−05 1102ARL4A 2.11255 0.000248586 31514 ST6GALNAC5 2.70116 0.00012729 29277RNF128 2.07512 8.41E−06 32854 TRIL 2.66182 5.76E−06 29124 RGS16 2.062377.60E−05 7617 GADD45G 2.62149 0.000385727 8211 GPRC5C 2.04839 5.12E−055349 DLL3 2.62131 0.000304562 26002 NGF 2.01869 9.29E−08 8399 GULP12.58556 2.34E−07 33375 UCP2 2.00568 0.000336807 16419 LOC283514 2.556547.18E−05 11039 HS.553187 −2.00024 0.000182185 12986 KCNS3 2.512253.59E−05 7972 GNAI1 −2.00345 0.000400577 6270 F3 2.50649 4.09E−09 1241ASTN1 −2.00503 0.000267753 32177 THBS4 2.48547 9.17E−05 29957 SEL1L3−2.00817 9.40E−06 6457 FAM181B 2.48092 0.0001071 9721 HS.348844 −2.067289.46E−05 12644 INPPL1 2.47363 7.08E−06 403 ADAMTS5 −2.09516 8.41E−0728624 PVRL3 2.47112 3.32E−05 5268 DIRAS2 −2.10442 0.000137286 2885C6ORF173 2.45642 9.25E−05 1042 ARHGAP22 −2.11082 9.20E−05 25218 MNX12.44999 9.15E−08 24440 MGC27121 −2.11477 1.75E−08 3835 CEBPD 2.447640.000160544 28124 PPP2R2C −2.12982 0.000152152 6197 ETS2 2.434822.74E−06 2812 C5ORF30 −2.14976 2.66E−06 8206 GPR98 2.37781 0.0004376368046 GOLSYN −2.15553 0.000225269 30664 SLC7A2 2.36837 5.80E−11 7626GAGE12C −2.16085 1.15E−07 153 ABAT −2.33538 2.64E−05 728 AMPH −2.191277.33E−05 28721 RAB3B −2.33887 0.000174687 28104 PPP1R1C −2.2262 3.10E−0731151 SNX10 −2.35266 0.000210353 28251 PRKACB −2.27738 6.28E−05 26936OSBPL8 −2.36481 0.000356878 26252 NR5A1 −2.3262 1.83E−08 30232 SHC4−2.36756 5.88E−06 5703 ECEL1 −2.83259 0.000107266 28689 RAB15 −2.377014.57E−05 16740 LOC387856 −2.84534 0.000116969 31222 SOX2OT −2.379295.44E−06 5631 DUSP6 −2.85825 8.35E−05 10203 HS.525171 −2.379490.000112764 31568 STC1 −2.8677 0.000365825 27287 PCSK1N −2.393960.000122619 227 ABLIM2 −2.8948 2.50E−06 30200 SH3BGRL2 −2.396960.000352607 27415 PELI2 −2.9135 2.07E−05 23918 LY6H −2.3986 0.0004264062538 C20ORF46 −2.92994 6.05E−05 31543 STAMBPL1 −2.40424 0.000210764 4639CSRNP3 −2.9364 6.80E−06 12256 HSPA12A −2.4161 5.00E−06 31628 STS-1−2.94215 9.63E−07 12209 HS3ST5 −2.42056 1.64E−08 30210 SH3GL3 −2.945590.000343973 1764 BTBD3 −2.43171 9.02E−05 10017 HS.452398 −3.017861.26E−05 32742 TOX2 −2.47449 6.41E−06 4705 CTNND2 −3.02031 4.91E−05 896AP1S2 −2.4814 0.000411095 24516 MGST1 −3.02712 1.36E−06 13764 LHFPL4−2.49494 0.000177306 4283 CNR1 −3.13389 0.000271812 12801 ITM2C −2.506350.000289334 13313 KLF6 −3.16185 3.73E−05 1641 BLCAP −2.52288 0.0003636487613 GAD1 −3.18763 0.000116615 30231 SHC3 −2.53075 2.70E−06 29400 RORB−3.1967 5.32E−08 33964 XPR1 −2.55331 2.67E−05 27566 PHYHIPL −3.210463.97E−05 28266 PRKCE −2.55567 1.75E−07 25628 MYO16 −3.21287 9.14E−0626521 OPTN −2.57271 0.000282119 25284 MPPED2 −3.23371 0.000114488 5325DKK3 −2.57389 8.27E−05 30531 SLC32A1 −3.23601 0.000270573 33325 UBL3−2.58024 0.000136606 29034 RELN −3.32027 1.95E−05 6859 FGF13 −2.584473.31E−05 26058 NKX2-1 −3.35392 4.84E−06 32210 THY1 −2.60259 0.00010704730114 SFRP1 −3.38198 0.00010787 30015 SEPT6 −2.63038 0.000101624 4965DBC1 −3.3928 1.25E−05 30351 SLC12A5 −2.64265 0.000195945 31763 SYT1−3.47973 0.00017165 607 AKR1C4 −2.66634 0.000217452 29624 RTN1 −3.487180.000208889 7635 GAGE2B −2.67196 4.87E−10 3121 CA10 −3.49466 0.00021747513177 KIAA1598 −2.67619 4.25E−05 24095 MAP4 −3.54296 0.000118613 1986C12ORF68 −2.67913 0.000114615 9476 HS.223856 −3.61087 5.18E−05 25516MTSS1 −2.69402 0.000290278 32343 TMEFF2 −3.6559 1.52E−06 32535 TMEM55A−2.70095 5.71E−06 713 AMER2 −3.65957 2.10E−08 25864 NDST3 −2.723449.29E−06 28417 PSD2 −3.70127 0.000123589 33591 VAT1 −2.74928 0.0002285557106 FLJ33590 −3.74166 4.19E−06 26498 ONECUT1 −2.77372 1.86E−05 4386COPG2IT1 −3.9994 7.98E−05 12634 INPP1 −2.79095 5.83E−05 3207 CALY−4.03219 3.21E−06 12954 KCNK12 −4.77483 2.25E−06 30824 SNCG −4.489562.76E−06 5352 DLX1 −4.89802 0.000316228 5455 DNER −4.58982 1.95E−0526332 NTS −4.92922 0.000411631 5550 DRD1IP −4.64486 5.89E−07 33565 UTS2−5.26698 2.91E−07 26253 NR5A2 −4.70942 1.65E−11 3220 CAMKV −5.355631.08E−05 2631 C22ORF42 −5.4488 2.15E−07 29220 RIT2 −5.49937 9.53E−1231359 SPOCK2 −5.66406 5.68E−06 12746 ISLR2 −5.6746 0.000247482 32591TMOD1 −5.689 7.66E−12 3215 CAMK2N1 −5.84508 2.30E−06 33592 VAT1L−6.26024 7.74E−07 27971 POMC −6.74106 6.72E−05 26219 NPTX2 −7.855111.15E−05 25939 NELL1 −8.58778 1.80E−08 33624 VGF −8.61516 1.37E−05 31223SOX3 −10.3701 8.71E−09 30314 SIX6 −12.5834 1.04E−12 26965 OTP −14.24696.08E−10 12743 ISL1 −21.0507 6.48E−05 31479 SST −21.3179 0.000281588

TABLE 6 shows an exemplary list of antibodies used as markers, includingconcentration (i.e. dilution) of antibodies used and exemplary sourcesof antibodies. In some embodiments, bound antibodies were identifiedwith any of Alexa488, Alexa555 and Alexa647-conjugated secondaryantibodies (Molecular Probes, Carlsbad, California). In someembodiments, biotinylated secondary antibodies were used to identify thebound primary antibodies followed by visualization via DAB(3,3′-Diaminobenzidine) chromogen. Antibody (primary) Dilution SourceLocation Human nuclear 1:100  Millipore Billerica, MA antigen Human cell1:100  Santa Cruz Santa Cruz, CA adhesion molecule Tyrosine1:1000/1:500  Pel-Freez/ Rogers, Hydroxylase ImmunoStar AR/Hudson, WI(TH) β-tubulin III  1:500/1:2000 Covance Littleton, CO/ Princeton, NJDoublecortin 1:100  Millipore Billerica, MA Human 1:300  R&DMinneapolis, specific MN Nestin Nestin # 130 1:50   R. McKay NINDS, NIHFoxA2 1:100  Santa Cruz Santa Cruz, CA Pitx3 1:100  Millipore Billerica,MA β-catenin 1:100  BD Franklin Lakes, NJ Collagen 1:100  OncogeneLaJolla, CA Cytokeratin 1:100  DAKO Glostrup DK Oct-4 1:200  Santa CruzBillerica, MA Ki-67 1:200/1:400 Zymed/DAKO San Francisco, CA GABA 1:2000Sigma St Louis, MI polyclonal Serotonin 1:2000 Sigma St Louis, MI (5-HT)GFAP 1:2000 DAKO Glostrup, DK polyclonal Calbindin 1:300  AbcamCambridge, MA VMAT2 1:200  Millipore Billerica, MA DAT 1:1000/1:2000Millipore Billerica, MA GFP 1:1000 Molecular Carlsbad, CA Probes Girk21:200  Abcam/ Cambridge MA/ Alomone Israel FoxG1 1:100  NeuraCellRensselaer, NY Pax6 1:100  Covance Princeton, NJ Otx2 1:2000 StrategicNewark, DE Diagnostics Lmx1a 1:2000 Millipore Pittsburgh, PA Synapsin1:1000 Sigma St Louis, MI Iba-1 1:200  Millipore Billerica, MA ED-11:200  Millipore Pittsburgh, PA Human NCAM 1:100  Santa Cruz Santa Cruz,CA (Eric-1) Human specific 1:1000 Stem Cells Inc. Newark, CA cytoplasm(SC-121) Nurr-1 1:1500 Perseus Japan Proteomics

TABLE 7 Exemplary contemplated differentiation into DA neurons by celltype limits. Cell type Marker Assay Description Proposed Limits MidbrainFOXA2/ IHC: Co-expression >50% FP/DA LMX1A @ day 13 & day 25; validationby qRT- PCR DA neuron FOXA2/TH; IHC: Co-expression >25% precursorTH/NURR1 @ day 25; validation by qRT- PCR Pluripotent cells OCT-4; IHC:@ day 13 &  <2% day 25; validation by qRT-PCR Proliferating cells Ki67IHC <25% (forebrain) FOXG1; IHC: @ day 13 & <10% precursor PAX6 day 25;validation by qRT-PCR DA neuron yield TH/FOXA2 IHC:: Co- >1DAn/hESCexpression @ day 25 out of hESC plated at day 0 In vivo survivalTH/FOXA2 Histology in vivo @ >2,000/animal (in vivo) 4 weeks aftergrafting of 200k cells

TABLE 8 Exemplary experiments to determine the role of certain factorsfor producing DA neurons. Dbc LDN SB PURM CHIR SHH BDNF GDNF AA AMPTGFB3 DAPT Control +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Complete+++ +++ +++ +++ dropout +/−SHH ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ +/−BDNF++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ +/−GDNF ++ ++ ++ ++ ++ ++ + ++ ++ ++ +++/−AA ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ +/−dbcAMP ++ ++ ++ ++ ++ ++ ++++ + ++ ++ +/−TGFB3 ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ +/−DAPT ++ ++ ++ ++++ ++ ++ ++ ++ ++ +

TABLE 9 Exemplary methods for scaling up mDA neuron culture, inparticular for use in producing GMP level cultures for use in theclinic. Intermediary scale Pilot scale (×3) (x ≥ 1) Clinical scale WCBthaw 1 vial - 1 × 10⁶ 7 vials - 7 × 10⁶ 42 vials - 7 × 10⁶ on 6 cm dishon 15 cm × 1 per 15 cm × 6 Passage 1 - 1:1-1:5 on 1:1-1:5 on 1:1-1:5 onclusters 6 cm dishes 15 cm dishes 15 cm dishes Passage 2 - 2 × 15 cmdishes 6 × 15 cm dishes 36 × 15 cm dishes clusters Passage 3 - 1 × 15 cmdish 3 × 15 cm dishes 18 × 15 cm dishes Accutase at high density at highdensity at high density (Neural (approximately (approximately(approximately induction) 3.0 × 10⁷ cells) 9.0 × 10⁷ cells) 5.4 × 10⁸cells) Passage 4 1:1 1:1 1:1 Accutase (Day 15-20) Cryo- approximatelyapproximately approximately preservation 1.2 × 10⁸, 3.6 × 10⁸, 2.16 ×10⁹, (Day 25) assume 50% assume 50% assume 50% loss after loss afterloss after cryopreservation = cryopreservation = cryopreservation =approximately approximately approximately 6 × 10⁷ 1.8 × 10⁸ 1 × 10⁹viable cells viable cells viable cells

TABLE 10 Exemplary in vivo assessment of hES line products- Graftcomposition. Assay Proposed Cell type Marker Description Limits DAneuron TH/FOXA2 Stereological >5,000 per yield assessment of 200,000total TH/FoxA2 grafted cell number (IHC) in grafts Proliferation Ki67(MIB-1) IHC for Ki67;   <1% Index percent of total cell numberPluripotent OCT-4/Nanog IHC <0.5% cells Serotonergic 5-HT (serotonin)IHC   <1% neurons Forebrain FOXG1; PAX6 IHC  <10% precursors TeratomaMyosin; IHC   <1% derivatives cytokeratins; αfetoprotein

TABLE 11 Exemplary in vivo assessment of hES line products- BehavioralAnalyses Proposed Test Parameter Assay Description Limits AmphetamineSum of Turning behavior <1 rotation/ rotations (*) rotations/min towardslesion side min (ipsi-minus following contralateral) intraperitonealamphetamine injection Stepping Test % Initiation of stepping 40-50%contralateral movement using the step limb contralateral to adjustments/lesion side total adjustments Cylinder test % use of Spontaneous Min.20% ipsilateral exploration with improvement limb/total ipsi vscontralateral vs. limb pre-grafting (*) In some embodiments, ratsexhibiting >6 rotations/min stably received grafts.

Experimental

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof. In the experimental disclosures which follow, thefollowing abbreviations apply: N (normal); M (molar); mM (millimolar);μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); pg (picograms); L and (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); U (units); min (minute); s and sec(second); deg (degree); pen (penicillin), strep (streptomycin) and ° C.(degrees Centigrade/Celsius).

Example I

Materials And Methods.

Methods Summary: Human ESC (H9, H1) and iPSC lines (2C6 and SeV6) weresubjected to a modified Dual SMAD-inhibition (Chambers, et al. Nat.Biotechnol. 27:275-280 (2009), herein incorporated by reference) basedfloor plate induction (Fasano, et al., Cell Stem Cell 6:336-347 (2010),herein incorporated by reference) protocol. Exposure to SHH C25II,Purmorphamine, FGF8 and CHIR99021 were optimized for midbrain floorplate and yield of novel populations of DA neuron (see FIG. 1d ).Following floor plate induction, further maturation (days 11-25 orlonger than 25 days in culture up to at least 100 days in culture) wascarried out in differentiation medium based on Neurobasal/B27 in thepresence of DA neuron survival and maturation factors (Perrier, et al.Proc Nall Acad Sci USA 101:12543-8 (2004), herein incorporated byreference) such as AA, BDNF, GDNF, TGFβ3 and dbcAMP (see full methodsfor details). The resulting DA neuron population were subjected toextensive phenotypic characterization via immunocytochemistry, qRT-PCR,global gene expression profiling, HPLC analysis for the detection ofdopamine and in vitro electrophysiological recordings. In vivo studieswere performed in hemiparkinsonian rodents (mouse or rats injected withthe 6OHDA toxin on one side of the animal's brain. The studies werecarried out inadult NOD-SCID IL2Rgc mice (Jackson labs) and adultSprague Dawley rats Taconic Farms, that received 6-hydroxydopaminelesions by stereotactic injections of the toxin as described previouslyas well as two adult rhesus monkeys that were treated with unilateralcarotid injections of MPTP. DA neurons were injected stereotactically inthe striata of the animals (150×10³ cells in mice, 250×10³ cells inrats) and a total of 7.5×10⁶ cells (distributed in 6 tracts; 3 on eachside of brain) in monkeys. Behavioral assays were performed at monthlyintervals post-grafting, including amphetamine mediated rotationalanalysis as well as a test for focal akinesia (“stepping test”) and limbuse (cylinder test). Rats and mice were sacrificed at 18-20 weeks andthe primates at 1 month post grafting. Characterization of the graftswas performed via stereological analyses of cell number and graftvolumes as well as a comprehensive phenotypic characterization viaimmunohistochemistry. Culture of undifferentiated human ES cells. hESClines H9 (WA-09, XX, passages 27-55 from when 10/2009), H1 (WA-01, XY,passages 30-40 from when 6/2010) and iPS cell lines 2C6 (Kim, et al.Cell Stem Cell 8:695-706 (2011), herein incorporated by reference) (XY,passages 20-30) and SeV6 (XY, passages 20-30; derived from MRC-5embryonic fibroblasts using non-integrating 4 factor Sendai vectorsystem (Ban, et al. Proc. Natl. Acad. Sci. U. S. A (2011) 108(34):14234-14239:10.1073/pnas.1103509108, herein incorporated by reference)were maintained on mouse embryonic fibroblasts at plating concentrationsestimated ranging from 0.5×10³ per cm² to 100×10³ per cm² based uponhuman ES cells which tend to cell cluster.(MEF, Global Stem, Rockville,Md.) in an optimal 20% knockout serum replacement (KSR, Invitrogen,Carlsbad, Calif.)-containing human ES cell medium (as describedpreviously (Kim, et al. Cell Stem Cell 8:695-706 (2011), hereinincorporated by reference). The use of knockout serum replacement mayrange from 0% to 40%.

Neural Induction. For floor plate-based midbrain dopamine neuroninduction, a modified version of the dual-SMAD inhibition (Chambers, etal. Nat. Biotechnol. 27:275-280 (2009), herein incorporated byreference) and floor plate induction (Fasano, et al. Cell Stem Cell6:336-347 (2010), herein incorporated by reference) protocol was usedbased on timed exposure to LDN-193189 (100 nM (ranging in concentrationfrom 0.5-50 μM, Stemgent, Cambridge, Mass.), SB431542 (10 μM (ranging inconcentration from 0.5-50 μM, Tocris, Ellisville, Mich.), SHH C25II (100ng/ml (ranging in concentration from 10-2000 ng/ml, R&D, Minneapolis,Minn.), Punnorphamine (2 μM (ranging in concentration from 10-500 ng/ml,Stemgent), FGF8 (100 ng/ml (ranging in concentration from 10-500 ng/ml,R&D) and CHIR99021 (CHIR; 3 μM (ranging in concentration from 0.1-10 μM,Stemgent). Note: for the floor plate induction protocol “SHH” treatmentrefers to exposure, i.e. contact, of cells to a combination of SHH C25II100 ng/ml+Purmorphamine (2 μM). Cells were plated (35-40×10³ cells/cm²)and cultured for 11 days on matrigel or geltrex (used as purchased) (BD,Franklin Lakes, N.J.) in Knockout serum replacement medium (KSR)containing DMEM, 15% knockout serum replacement, 2 mM L-glutamine and10-μM (ranging in concentration from 1-25 μM β-mercaptoethanol. KSRmedium was gradually shifted to N2 medium starting on day 5 ofdifferentiation, by mixing in ratios of 75% (KSR):25% (N2) on day 5-6,50% (KSR):50% (N2) day 7-8 and 25% (KSR):75% (N2) on day 9-10, asdescribed previously (Chambers, et al. Nat. Biotechnol. 27:275-280(2009), herein incorporated by reference). On day 11, media was changedto Neurobasal medium/B27medium (1:50 dilution)/L-Glut (effective ranges0.2-2 mM)) containing medium (NB/B27; Invitrogen) supplemented with CHIR(until day 13) and with BDNF (brain-derived neurotrophic factor, 20ng/ml ranging from 5 to 100; R&D), ascorbic acid (AA; 0.2 mM (ranging inconcentration from 0.01-1 mM), Sigma, St Louis, Mich.), GDNF (glial cellline-derived neurotrophic factor, 20 ng/ml (ranging in concentrationfrom 1-200 ng/ml); R&D), TGFβ3 (transforming growth factor type ß3, 1ng/ml (ranging in concentration from 0.1-25 ng/ml); R&D), dibutyryl cAMP(0.5 mM (ranging in concentration from _0.05-2 mM); Sigma), and DAPT (10nM (ranging in concentration from 0.5-50 nM); Tocris,) for 9 days. Onday 20, cells were dissociated using Accutase (Innovative CellTechnology, San Diego, Calif.) and replated under high cell densityconditions (for example from 300-400 k cells/cm²) on dishes pre-coatedwith polyornithine (PO); 15 μg/ml (ranging in concentration from 1-50μg/ml)/Laminin (1 μg/ml) (ranging in concentration from 0.1-10μg/ml)/Fibronectin (2 μg/ml (ranging in concentration from 0.1-20 μg/ml)in differentiation medium (NB/B27+BDNF, AA, GDNF, dbcAMP (ranging inconcentration as described herein), TGFβ3 and DAPT (ranging inconcentration as described herein) until the desired maturation stagefor a given experiment.

For rosette-based DA neuron induction previously described protocolswere followed in part (Perrier, et al. Proc Natl. Acad Sci USA101:12543-8 (2004), herein incoropoated by reference) with at least oneexception where dual-SMAD inhibition was used to accelerate the initialneural induction step. In brief, hESCs were induced towards neural fateby coculture with irradiated MS5 cells in KSR supplemented with SB431542and Noggin (250 ng/ml (ranging in concentration from 10-1000 ng/ml);R&D), from day 2-8 and SHH+FGF8 from day 6-11 of differentiation. After11 days in KSR, neural rosettes were manually isolated and cultured (P1stage) in N2 medium supplemented with SHH, FGF8, BDNF and AA asdescribed previously (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8(2004), herein incorporated by reference). After 5-7 days in P1 stage,rosettes were again harvested mechanically and triturated followingincubation in Ca²/Mg²-free Hanks' balanced salt solution (HBSS) for 1 hand replated on polyornithine (PO)/Larninin/Fibronectin coated plates.Patterning with SHH/FGF8 was continued for 7 days at P2 stage followedby final differentiation in the presence of BDNF, AA, GDNF, TGFb3 anddbcAMP as described above until the desired maturation stage for a givenexperiment (typically 5-7 days for transplantation studies or 32 daysfor in vitro functional studies).

Gene expression analyses. Total RNA was extracted during differentiationat days: 0, 1, 3, 5, 7, 9, 11, 13 and 25 from each condition of controlLSB, LSB/SHH/FGF8 and LSB/SHH/FGF8/CHIR using the RNeasy kit (Qiagen,Valencia, Calif.). For microarray analysis, total RNA was processed bythe MSKCC Genomic core facility and hybridized on Illumina Human ref-12bead arrays according to the specifications of the manufacturer.Comparisons were performed among each days and conditions using theLIMMA package from Bioconductor (worldwideweb.bioconductor.org). Genesfound to have an adjusted P-value <0.05 and a fold change greater thantwo were considered significant. Some of the descriptive microarray dataanalyses and presentation was performed using a commercially availablesoftware package (Partek Genomics Suite (version 6.10.0915)). ForqRT-PCR analyses, total RNA at day 25 of each condition was reversetranscribed (Quantitech, Qiagen) and amplified material was detectedusing commercially available Taqman gene expression assays (AppliedBiosystems, Carlsbad, Calif.) with the data normalized to HPRT. Eachdata point represents 9 technical replicates from 3 independentbiological samples. Raw data of microarray studies are not yet availableat GEO worldwideweb.ncbi.nlm.nih.gov/geo). Animal Surgery. Rodent andmonkey procedures were performed following NIH guidelines, and wereapproved by the local Institutional Animal Care and Use Committee(IACUC), the Institutional Biosafety Committee (IBC) as well as theEmbryonic Stem Cell Research Committee (ESCRO).Mice. NOD-SCID IL2Rgc null mice (20-35 g in weight; Jackson Laboratory,Bar Harbor, Me.) were anesthetized with Ketamine (90 mg/kg; Akorn,Decatur, Ill.) and Xylazine (4 mg/kg Fort Dodge, Iowa).6-hydroxydopamine (10 μg (ranging in concentration from 0.1-20 μg)6-OHDA (Sigma-Aldrich) was injected stereotactically into the striatumat the following coordinates (in millimeters): AP, 0.5 (from bregma; askull suture used as reference for stereotactic surgery); ML, −2.0; DV,−3.0 (from dura a membrane covering the brain used for reference). Micewith successful lesions (an average of >6 rotations/minutes) wereselected for transplantation. A total of 150×10³ cells were injected ina volume of 1.5μl into the striatum at the following coordinates (inmm): AP, 0.5; ML, −1.8; DV, 3.2. The mice were sacrificed 18 weeks posttransplantation.Rats. Adult female Sprague-Dawley (Taconic, Hudson, N.Y.) rats (180-230g) were anesthetized with Ketamine (90 mg/kg) and xylazine (4 mg/kg)during surgical procedures. Unilateral, medial forebrain bundle lesionsof the nigro-striatal pathway were established by stereotaxic injectionof 6-OHDA (3.6 mg/ml in 0.2% ascorbic acid and 0.9% saline, Sigma) attwo sites (Studer, et al. Nature Neurosci. 1:290-295 (1998), hereinincorporated by reference). Rats were selected for transplantation ifamphetamine-induced rotation exceeded 6 rotations/min by 6-8 weeks postinjection. 250×103 cells were transplanted into the striatum of eachanimal (Coordinates: AP+1.0 mm, ML −2.5 mm and V-4.7 mm; toothbar set at−2.5). Control rats received PBS instead. The surgical procedures weredescribed previously (Studer, et al. Nature Neurosci. 1:290-295 (1998),herein incorporated by reference). Daily intraperitoneal injections ofcyclosporine 15 mg/kg (Bedford. Labs, Bedford, Ohio) were started 24hours prior to cell grafting and continued until sacrifice, 20 weeksfollowing cell grafting.Primates. Two adult (17-18 yr old; 10-12 kg; female) rhesus monkeys wererendered hemiparkinsonian via carotid MPTP administration followed byweekly I.V. MPTP administration to create a bilateral parkinsoniansyndrome (Kordower, et al. Science 290:767-773 (2000), hereinincorporated by reference). Both animals displayed parkinsonian symptomsconsistent with a moderately-severe lesion based on behavioral analysisincluding crooked posture, dragging of leg and symptoms of rigor(inflexibility of movement), neglect (motor awareness to lateralizedstimulus) and bradykinesia (slow movement intiation). These parameterscan be assessed in monkeys using a modified parkinsonian clinical ratingscale (CRS). On the day of transplantation surgery, animals weretranquilized with ketamine (3.0 mg/kg, IM) and dexdomitor (0.02-0.04mg/kg IM), intubated to maintain a stable airway and anesthetized withisoflurane. They were then placed into a stereotaxic frame for surgery.Both rhesus monkeys underwent a single surgery with three intracranialinjections of human floor plate-derived DA cultures based on stereotaxiccoordinates (Paxinos, et al. The Rhesus Monkey Brain in StereotaxicCoordinates (Academic Press, 2000), herein incorporated by reference).Bilateral injections of cells (10 ul/injection; 125,000 cell/up wereperformed at three sites (1-posterior caudate, 2-pre-commissural putamenand overlying white matter) for a total volume of 30 μl per hemisphere.An infusion pump attached to a stereotaxic micromanipulator was utilizedto deliver the cells at a rate of though a 500 Hamilton syringe with 28G needle. After the injections were completed, the needle was left inplace for an additional 2-5 minutes to allow the infusate to diffuse offthe needle tip before slowly retracting the syringe. Immediatelyfollowing surgery, the animals received analgesics (buprenex, 0.01 mg/kgIM, BID for 72 hours post surgery; meloxicam, 0.1 mg/kg SQ, SID for 72hours post surgery) as well as an antibiotic (cephazolin, 25 mg/kg IM,BID) until 72-hours post-surgery. The animals received cyclosporine A(Neoral, Sandimmune) orally (30 mg/kg tapered to 15 mg/kg) once dailybeginning 48-hrs prior to surgery until sacrifice, one month followingtransplantation.Behavioral Assays. Amphetamine-induced rotations (mice and rats) and thestepping test (rat) were carried out before transplantation and 4, 8,12, 18 weeks after transplantation. Rotation behavior in mice wasrecorded 10 min after i.p. injection of d-amphetamine (10 mg/kg, Sigma)and recorded for 30 minutes. Rotation behavior in rats was recorded 40min after i.p. injection of d-amphetamine (5 mg/kg) and automaticallyassessed by the TSE VideoMot2 system (Geiritany). The data werepresented as the average number of rotations per minute. The steppingtest was modified from Blume, et al. Exp. Neurol. 219:208-211 (2009) andCrawley, et al. What's Wrong With My Mouse: Behavioral Phenotyping ofTransgenic and Knockout Mice (Wiley-Liss, 2000), all of which are hereinincorporated by reference. In brief, each rat was placed on a flatsurface; its hind legs were lifted by gently holding up the tail toallow the forepaws alone to touch the table. The experimenter pulled therat backwards 1 meter at a steady pace. Adjusting step numbers from bothcontralateral and ipsilateral forepaws were counted. Data was presentedas the percentage of contralateral/(contralateral+ipsilateral) adjustingsteps. The cylinder test was performed by placing each animal in a glasscylinder and counting the number of ipsilateral versus contralateral pawtouches (out of 20 touches) to the wall of the cylinder as describedpreviously (Tabar, et al. Nature Med. 14:379-381 (2008), hereinincorporated by reference). Tissue Processing. Mice and Rats: Animals(mice and rats) received overdoses of Pentobarbital intraperitoneally(50 mg/kg) to induce deep anesthesia and were perfused in 4%paraformaldehyde (PFA). Brains were extracted, post-fixed in 4% PFA thensoaked in 30% sucrose solutions for 2-5 days. They were sectioned on acryostat after embedding in O.C.T. compound (Sakura-Finetek, Torrance,Calif.).Primates: Animals were sacrificed under deep anesthesia with ketamine(10 mg/kg, Intramuscular (IM)) and pentobarbital (25 mg/kg, intravenous(IV)) via cardiac perfusion with heparinized 0.9% saline followed byfresh cold 4% PFA fixative (pH7.4). Immediately following primaryfixation, brains were removed from the skull and post-fixed in 4% PFA,free-floating, for 24-36 hrs. They were then rinsed and re-suspended in10% sucrose on a slow shaker at 4° C., and allowed to “sink”. Theprocess was then repeated in 20% sucrose followed by 30% sucrose. Wholebrains were cut coronally into 40 um serial sections on a frozen sledgemicrotome and stored free-floating in cryopreservative medium at −20°Celcius.Immunohistochemistry: Cells were fixed in 4% PFA and blocked with 1%bovine serum albumin (BSA) with 0.3% Triton. Brain tissue sections werewashed in cold PBS and processed similarly. Primary antibodies werediluted in 1-5% BSA or Normal Goat Serum and incubated according tomanufacturer recommendations. A comprehensive list of antibodies andsources is provided as Table 6. Appropriate Alexa488, Alexa555 andAlexa647-conjugated secondary antibodies (Molecular Probes, Carlsbad,Calif.) were used with 4′,6-diamidino-2-phenylindole (DAPI) nuclearcounterstain (Thermo Fisher, Rockford, Ill.). For some analysesbiotinylated secondary antibodies were used followed by visualizationvia DAB (3,3′-Diaminobenzidine) chromogen. HPLCAnalysis. Reversed-phase HPLC with electrochemical detection formeasuring levels of dopamine, Homovanillic acid (HVA) and DOPAC(3,4-Dihydroxy-Phenylacetic Acid) was performed as described previously(Roy, et al. Nature Med. 12:1259-1268 (2006); Studer, et al. Brain Res.Bull. 41:143-150 (1996), all of which are herein incorporated byreference). Culture samples were collected in perchloric acid at day 65of differentiation. For some experiments DA was measured directly in themedium using the same detection system but following aluminum extractionof dopamine and its metabolites using a commercially available kit asdescribed previously (Studer, et al. Brain Res. Bull. 41:143-150 (1996),herein incorporated by reference). Electrophysiological recordings:Cultures were transferred to a recording chamber on an uprightmicroscope equipped with a 40× water-immersion objective (EclipseE600FN; Nikon); cultures were perfused with saline containing in mM: 125NaCl, 2.5 KCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl, 1 MgCl₂, and 25 glucose(34° C.; saturated with 95% O₂-5% CO₂; pH 7.4; 298 mOsm/L). The salineflow rate was 2-3 ml/min running through an in-line heater (SH-27B withTC-324B controller; Warner Instruments). Neurons were visualized byvideo microscopy with a cooled-CCD digital camera (CoolSNAP ES²,Photometrics, Roper Scientific, Tucson, Ariz.). Cells selected forelectrophysiological recordings had neuron-like shapes with finebranching neurites. Somatic whole-cell patch-clamp recordings in currentclamp configuration were performed with a MultiClamp 700B amplifier(Molecular Devices). Signals were filtered at 1-4 kllz and digitized at5-20 kHz with a Digidata 1440A (Molecular Devices). Recording patchelectrodes were fabricated from filamented horosilicate glass (SutterInstruments) pulled on a Flaming-Brown puller (P-97, Sutter Instruments)and had resistances of 4-6 MΩ. in the bath. Electrodes were filled withinternal solution containing in mM: 135 K-MeSO₄, 5 KCl, 5 HEPES, 0.25EGTA, 10 phosphocroeatine-di(tris), 2 ATP-Mg, and 0.5 GTP-Na (pH 7.3,osmolarity adjusted to 290-300 mOsm/L). The amplifier bridge circuit wasadjusted to compensate for electrode resistance and monitored. Electrodecapacitance was compensated. When series resistance increased >20%during the recording, the data were discarded because increasedresistance suggested a partial technical failure during recordings. CellCounts and Stereological Analyses. The percentages of marker positivecells at the floor plate (day 11) FIG. 1, midbrain dopamine neuronprecursor (day 25), FIG. 2 and mature DA neuron stages (day 50 or later)FIGS. 3 and 11, were determined in samples derived from 3 independentexperiments each. Images for quantification were selected in a uniformrandom manner and each image was scored first for the number ofDAPI-positive nuclei, followed by counting the number of cellsexpressing the marker of interest. Data are presented as mean±SEM.Quantification of human cells (identified with anti-hNA) and TH+ neuronswithin grafts was perfoinied on every tenth section where a graft wasidentifiable. Cell counts and graft volume was determined using theoptical fractionator's probe and the Cavalieri estimator using theStereo Investigator software (MBF bioscience, Vermont) as describedpreviously in Tabar, et al. Nat. Biotechnol. 23:601-606 (2005), hereinincorporated by reference. Data are presented as estimated total cellnumber and total graft volume+/−standard error of means (SEM).

The following formulations describe exemplary cell culture medium foruse in developing embodiments of the present inventions.

hESC medium for maintenance (1 liter): 800 mL DMEM/F12, 200 mL ofKnockout Serum Replacement, 5 mL of 200 mM L-Glutamine, 5 mL ofPen/Strep, 10 mL of 10 mM MEM minimum non-essential amino 15 acidssolution, 55 μM of 13-mercaptoethanol, and bFGF (final concentration is4 ng/mL).KSR medium for hESC differentiation (1 liter): 820 mL of Knock out DMEM,150 mL of Knock out Serum Replacement, 10 mL of 200 mM L-Glutamine, 10mL of Pen/Strep, 10 mL of 10 mM MEM, and 55 μM of 13-mercaptoethanol.N2 medium for hESC differentiation (1 liter): 985 ml dist. H₂0 withDMEM/F12 powder, 1.55 g of glucose (Sigma, cat. no. G7021), 2.00 g ofsodium bicarbonate (Sigma, cat. no. S5761), putrescine (100 uL aliquotof 1.61 g dissolved in 100 mL of distilled water; Sigma, cat. no.P5780), progesterone (20 uL aliquot of 0.032 g dissolved in 100 mL 100%ethanol; Sigma, cat. no. P8783), sodium selenite (60 uL aliquot of 0.5mM solution in distilled water; Bioshop Canada, cat. no. SEL888), and100 mg of transferrin (Celliance/Millipore, cat. no. 4452-01), and 25 mgof insulin (Sigma, cat. no. 16634) in 10 mL of 5 mM NaOH.Dulbecco's Modification of Eagles Medium (DMEM), with 10% FBS forpreparing PMEF ((primary mouse embryo fibroblast (PMEF)) feeder cells)(1 liter): 885 mL of DMEM, 100 mL of FBS, 10 mL of Pen/Strep, and 5 mLof L-Glutamine.Alpha Minimum Essential Medium (MEM) with 10% FBS for preparing MS-5feeder cell medium (1 liter): 890 mL of Alpha MEM, 100 mL of FBS, 10 mLof Pen/Strep Gelatin solution (500 ml): Dissolve 0.5 g of gelatin in 500ml of warm (50-60° C.) Milli-Q water. Cool to room temperature.

Example II

This example describes the discovery of small molecules and contacttiming for providing directed differentiation of FOXA2+LMX1A+DA neuronsof the present inventions.

The following is a brief summary of some of the experimental discoveriesdescribed herein: Treatment of Dual-SMAD inhibited cells with SHHagonists (purmorphamine+SHH) and FGF8 (S/F8) in the absence of CHIR99021showed significantly lower expression of FOXA2 by day 11 and completelack of LMX1A expression (FIG. 1a,b ). The anterior marker OTX2 wasrobustly induced in LSB and LSB/S/F8/CHIR treated cultures, but notunder LSB/S/F8 conditions (FIG. 1a,c ). The inventors previous usedseveral other directed differentiation methods that resulted in cellpopulations containing DA-like neurons. These DA-like neurons were usedin transplantation studies that resulted in concerns on the further useof these cells for therapeutic applications. For examples, proceduresdescribed in Perrier et al., 2004 and Fasano et al., 2010, including MS5neural induction, resulted in rosette cell formation and were used tomake Day 11 precursors, see FIGS. 2, 16 and 17 for examples, that werefurther used to derive DA-like neurons. These neurons resulted from alow percentage of the precursor cells in the resulting Day 11 cellpopulations. Transplantation studies that used these neurons showed poorpost transplant viability and loss of the DA-like neuronal phenotype inaddition to observations of post transplantation development ofinappropriate neural types along with loss of growth control, which ledto development of teratomas. See FIGS. 16 and 17.

Specifically, at PO hESCs were contacted with molecules for beginningneural induction of Oct4+ cells into rosette cells using MS5 feedercells (Perrier et al., 2004). At the P1 stage Rosette cells wereexpanded by contacting cells with additional molecules fordifferentiating cells into cells at stage P2 with specific expressionpatterns including Pax2+/En1+DA progenitor cells and were furtherdifferentiated into TH+/En1+DA neurons. These cells were used forengraftment in 6OHDA lesioned rats, immunosuppressed with Cyclosporin A.Those transplantation studies showed poor in vivo viability, loss of theTH+ phenotype, concerns about further growth into unwanted, possiblylethal, cells, i.e. teratomas, and growth of cells into inappropriateneural types that would cause additional medical problems for thepatient.

There were very small numbers of surviving TH+ neuron at 4.5 moths aftertransplantation (<50 TH+ cells/animal) in grafts from rosette derived DAneuron precursors FIG. 16A. However, in contrast to TH+ cells, GFPmarked cells (GFP was driven by a ubiquitous promomoter) did survivequite well after transplantation. This suggests that most survivingcells following transplantation were neural cells of non-DA neuronidentity (16B). Few graft-derived cells (hNA+(green) co-express TH (red)again suggesting that most grafted human cells adopt a non-DA neuronphenotype FIG. 16C. Panels 16 D-E show that D-E, despite the very poorin vivo survival there was some (low and highly variable) improvement ina few behavioral assays such as amphetamine induced rotations (D),cylinder test and spontaneous rotations (E). Feeder-free neuralinduction was carried out as previously described (Chambers et al.,2009) but further modified to yield floor plate cells (Fasano et al.,2010). In the modified Dual-SMAD inhibition method for differentiatingpluripotent cells into floor plate cells, the inventors' previouslydiscovered that high concentrations of SHH were required for FPinduction by day 11. For example, in some embodiments, Sonic C25II wasadded at 200 ng/ml. In some experiments, DKK-1 (R&D; 100 ng/ml) FGF8(R&D; 50 ng/ml), Wnt-1 (Peprotech; 50 ng/ml) and retinoic acid (R&D; 1mM) were added, See FIG. 17. However none of the resulting cellpopulations at day 11 using previous methods, contained the highpercentage of of FOXA2+/LMX1A+ midbrain floor-plate progenitor cellsusing methods of the present inventions.

As shown herein, a cell population containing pluripotent cells waschosen by the inventors for a starting population and plated at Day 0.Cell are grown to near confluency prior to differentiation (between60-100% confluence). These cells were contacted with Dual SMADinhibitors (i.e. exposure to LDN-193189+SB431542=“LSB”) on Day 0. Theinventors followed a cell population with regular feedings containingfresh LSB until Day 11 and discovered that some remaining cells wereLMX1A+ but did not express FOXA2 (FIG. 1a,b ). The inventors platedduplicate starting cell populations then tested for cell types (i.e.gene/protein expression patterns) after contacting with mixturescontaining any of the following SHH agonists (purmorphamine+SHH) andFGF8 (S/F8) contacting the cells with different exposure regimens, i.e.contacting cells at Day 0, or Day 1, or Day 2, etc. for specific amountsof time, i.e. 24 hours, 48 hours, etc. Three primary exemplary cultureconditions tested were 1) cells contacted with LDN/SB (LSB) on Day 0then contacted with fresh LSB until Day 5, on Day 5 cells were contactedwith fresh LDN without SB until Day 11, 2) cells contacted with LDN/SB(LSB) on Day 0 then contacted with fresh LSB until Day 5, on Day 5 cellswere contacted with fresh LDN without SB until Day 11 while during thistime cells were additionally contacted with fresh purmorphamine, SHH andFGF8 until Day 7 and 3) cells contacted with LDN/SB (LSB) on Day 0 thencontacted with fresh LSB until Day 5, on Day 5 cells were contacted withfresh LDN without SB until Day 11 while during this time cells wereadditionally contacted with fresh purmorphamine, SHH and FGF8 until Day7 while additionally contacted with fresh CHIR starting on Day 3 ofculture until Day 11 with several variations of these primary conditionsin order to determine optimal yield of cell types. Systematiccomparisons of the three culture conditions (FIG. 1d ) were performedusing global temporal gene expression profiling. See exemplary FIG. 8and Tables 1-6. Hierarchical clustering of differentially expressedgenes segregated the three treatment conditions by day 11 ofdifferentiation (FIG. 8a ). FOXA1, FOXA2 and several other SHHdownstream targets including PTCH1 were amongst the most differentiallyregulated transcripts in LSB/S/F8/CHIR versus LSB treatment sets (FIG.1e ).Expression of LMXIA, NGN2, and DDC indicated establishment ofmidbrain DA neuron precursor fate already by day 11 (FIG. 1e,f ). Incontrast, LSB cultures by day 11 were enriched for dorsal forebrainprecursor markers such as HES5, PAX6, LHX2, and EMX2. Direct comparisonof LSB/S/F8/CHIR versus LSB/S/F8 treatment (FIG. 1f ) confirmedselective enrichment for midbrain DA precursor markers in LSB/S/F8/CHIRgroup and suggested hypothalamic precursor identity in LSB/S/F8 treatedcultures based on the differential expression of RAX1, SIX3, and SIX6(see also POMC, DTP expression in FIG. 2d ).

Exemplary lists of differentially expressed transcripts for day 11 areshown in Tables 1, 2 and day 25 in Tables 3-5 and gene ontology analysisFIG. 8b (DAVID; http://david.abcc.ncifcrf.gov) confirmed enrichment forcanonical WNT signaling upon CHIR treatment. Raw data are not yetavailable at GEO worldwideweb.ncbi.nlm.nih.gov/geo/accession#: [TBD]).Comparative temporal analysis of gene expression for midbrain DAprecursor markers (FIG. 1g ) versus markers of anterior and ventralnon-DA neuron fates (FIG. 1h ) partitioned the three inductionconditions into: i) LSB: dorsal forebrain; ii) LSB/S/F8:ventral/hypothalamic and iii) LSB/S/F8/CHIR: midbrain DA precursoridentity.

Example III

Differentiation of DA neurons. For further differentiation, precursorcells were maintained in. a medium promoting neuronal maturation(BAGCT—see material and methods). The following types of comparisonswere made between the populations of differentiated cells resulting fromprevious methods and methods of the present inventions: A)Immunocytochemical analysis at day 50 of differentiation for TH incombination with LMX1A, FOXA2 and NURR1, B) Quantification of TH+,FOXA2+, LMX1+, and NURR1+ cells out of total cells comparingrosette-derived versus floor plate-derived (LSB/S/F8/CHIR) cultures. C)Quantification of the percentages of serotonin+(5-HT), and GABA+neuronal subtypes (non-DA neuron contaminants) at day 50 in floor plateand rosette-derived DA neuron cultures. And D) HPLC analysis formeasuring dopamine and metabolites: Comparison of the DA, DOPAC and HVAlevels between floor plate versus rosette-derived cultures. By day 25,three precursor cell populations yielded Tuj1+ neurons (FIG. 2a ) andcells expressing TH, the rate-limiting enzyme in the synthesis of DA.However, LSB/S/F8/CHIR treatment yielded TH+ cells that co-expressedLMX1A and FOXA2 and displayed strong induction of the nuclear receptorNURR1 (NR4A2) (FIG. 2a,b ). Comparing gene expression in day 13 versusday 25 cultures confirmed robust induction of other postmitotic DAneuron markers (FIG. 2c ). Characterizing DA neuron identity at day 25in comparison to LSB and LSB/S/F8 treated cultures confirmed enrichmentfor known midbrain DA neuron transcripts and identified multiple novelcandidate markers (FIG. 2d , Tables 3-5, FIG. 8b ). For example, thetranscript most highly enriched in LSB/S/F8/CHIR (midbrain DA group) wasTTF3, a gene not previously associated with midbrain DA neurondevelopment, but highly expressed in the human substantia nigra (FIG. 8c; Allen Brain Atlas: http://human.brain-map.org).

Similar data were obtained for EBF-1, EBF-3 (FIG. 8c ) as well as TTR, aknown transcriptional target of FOXA2 in the liver. The data obtainedduring the development of the present inventions indicated enrichment ofseveral PITX genes in midbrain DA precursor cells. PITX3, a classicmarker of midbrain DA neurons, was also robustly expressed at day 25 ofdifferentiation (FIG. 2e ). Finally, both midbrain floor plate and DAneuron induction could be readily reproduced in independent hESC andhiPSC lines (FIG. 9). The data demonstrated herein showed that theLSB/S/F8/CHIR protocol as opposed to other tested protocols yields cellsexpressing a marker profile matching midbrain DA neuron fate.

In vitro and in vivo properties of floor plate-derived DA neurons werecompared to DA-like neurons obtained via a neural rosette intermediate(FIGS. 10 and 16). Patterning of neural rosettes represents thecurrently most widely used strategy for deriving DA neurons from hPSCs.Both floor plate- and rosette-based protocols were efficient atgenerating TH+ neurons capable of long-term in vitro survival (day 50 ofdifferentiation; FIG. 3a ). However, the percentage of TH+ cells wassignificantly higher in floor plate-derived cultures (FIG. 3b ). WhileTH+ cells in both protocols displayed co-expression of NURR1, floorplate-derived DA neurons co-expressed FOXA2 and LMX1A (FIG. 3a,b ). FewGABA and serotonin (5-HT)-positive neurons were observed (FIG. 3c ). DA,and its metabolites DOPAC and HVA, were present in cultures generatedwith either protocol, but DA levels were approximately 8 times higher infloor plate cultures (FIG. 3d,e ). Midbrain DA neurons exhibitedextensive fiber outgrowth and robust expression of mature neuronalmarkers including synapsin, dopamine transporter (DAT), and G-proteincoupled, inwardly rectifying potassium channel (Kir3.2—also calledGIRK2—expressed in substantia nigra pars compacta (SNpc) DA neurons)(FIG. 3f , FIG. 11). SNpc DA neurons in vivo exhibit anelectrophysiological phenotype that differentiates them from most otherneurons in the brain. In particular, they spike spontaneously at a slow(1-3 Hz) rate. Moreover, this slow spiking is accompanied by a slow,sub-threshold oscillatory potential. After 2-3 weeks in vitro, thesesame physiological features are displayed by SNpc DA neurons culturedfrom early postnatal mice. The DA neurons differentiated from hESCsconsistently (4/4) displayed this distinctive physiological phenotype(FIG. 3g-i ).

Maintainence of mDA neurons in vitro at d65 showed TH positive neuronsare still expressing FoxA2 and extend long fibers typical for mDAneurons. FIG. 3A. DA release measurement by HPLC showed d65 old TH+neurons are functional in vitro FIG. 3B.

Example IV

Engraftment of novel DA neuronal cell population in rodents, i.e. miceand rats containing damaged neurons.

One of the challenges in the field is the ability to generatehPSC-derived midbrain DA neurons that functionally engraft in vivowithout the risk of neural overgrowth or inappropriate differentiationinto non-midbrain neurons or develop teratomas. Based on fetal tissuetransplantation studies, the inventors' contemplated that the time ofcell cycle exit, marked by expression of NURR1, may be a suitable stagefor grafting (approximately day 25 of differentiation—FIG. 2). Initialstudies using day 25 cells in non-lesioned adult mice showed robustsurvival of hPSC-derived FOXA2+/TH+ neurons at 6 weeks aftertransplantation (FIG. 12). Survival of FOXA2+/TH+ cells long-term inParkinsonian hosts without resulting in neural overgrowth was tested. Tothis end, 6-hydroxy-dopamine (6-OHDA) lesions (Tabar, et al. Nature Med.14:379-381 (2008), herein incorporated by reference) were made inNOD-SCID IL2Rgc null mice, a strain that efficiently supports xenograftsurvival with particular sensitivity for exposing rare tumorigenic cells(Quintana, et al. Efficient tumour formation by single human melanomacells. Nature 456:593-598 (2008), herein incorporated by reference).Both floor plate- and rosette-derived DA neuron cultures were grafted(150×10³/animal) without prior purification in order to reveal potentialcontaminating cells with high proliferative potential. Four and a halfmonths after transplantation floor plate-derived DA neuron grafts showeda well-defined graft core composed of TH+ cells co-expressing FOXA2 andthe human specific marker hNCAM (FIG. 4a-e ). Functional analysis showeda complete rescue of amphetamine-induced rotation behavior. In contrast,rosette-derived neuronal grafts showed few TH+ neurons, did not producea significant reduction in rotation behavior (FIG. 4d ) and displayedmassive neural overgrowth (graft volume >20 mm³; FIG. 13). Extensiveovergrowth of rosette-derived neuronal cells used in grafting asreported herein was comparable to previous work with rosette-derived DAgrafts from the inventors' group (Kim, et al. miR-371-3 ExpressionPredicts Neural Differentiation Propensity in Human Pluripotent StemCells. Cell Stem Cell 8:695-706 (2011), herein incorporated byreference) and others (Hargus, et al. Proceedings of the NationalAcademy of Sciences of the United States of America 107:15921-15926(2010), herein incorporated by reference). The overgrowth was likely dueto the longer survival periods (4.5 months versus 6 weeks), lack of FACSpurification prior to transplantation and choice of NOD-SCID IL2Rgc nullhost. The number of proliferating Ki67+ cells was minimal in floorplate-derived grafts (<1% of total cells), while rosette-derived graftsretained pockets of proliferating neural precursors. Neural overgrowthis thought to be caused by primitive anterior neuroectodermal cellswithin the graft (Elkabetz, et al. Genes Dev. 22:152-165 (2008); Aubry,et al. Proc. Natl. Acad. Sci. USA 105:16707-16712 (2008), hereinincorporated by reference). This hypothesis was supported by theexpression of the forebrain marker FOXG1 in rosette-but not floorplate-derived grafts. A small percentage of astroglial cells werepresent in both floor plate- and rosette-derived grafts, though mostGFAP+ cells were negative for human markers indicating host origin (FIG.13).

Results in NOD-SCID IL2Rgc null mice described herein demonstratedrobust long-term survival of FOXA2+/TH+ neurons, complete reversal ofamphetamine-induced rotation behavior and lack of any signs of neuralovergrowth. However, some of these outcomes could be attributable to thespecific use of NOD-SCID IL2Rgc null mice. To test this hypothesis,floor plate-derived DA neuron cultures (250×10³ cells) were transplantedin adult 6-OHDA lesioned rats immunosuppressed pharmacologically usingcyclosporine A. Five months after transplantation graft survival wasrobust (FIG. 4e-h ) with an average of more than 15,000 TH+ cellsco-expressing FOXA2 (FIG. 4g ), and human nuclear antigen (hNA) (FIG. 4e); TH+/hNCAM+ fibers emanated from the graft core into the surroundinghost striatum (FIG. 4f ). In addition to FOXA2, TH+ cells expressedmidbrain DA neuron markers PTTX3 and NURR1 (FIG. 4h-j ). Behavioralanalyses showed complete rescue of amphetamine-induced rotationalasymmetry, in contrast to sham-grafted animals that did not showimprovements (FIG. 4k ). Grafted animals also showed improvements in thestepping test (FIG. 4l ) measuring forelimb akinesia and in the cylindertest (FIG. 4m ), assays that do not depend on pharmacologicalstimulation of the DA system. The late onset of recovery (approximately3-4 months after transplantation) is expected for human DA neurons anddepends on the rate of in vivo maturation such as the levels of DATexpression (FIG. 4n ). The presence of TH+ cells expressing Kir3.2channels (GIRK2) or calbindin indicate that both SNpc (A9) and ventraltegmental area (A10) DA neurons are present in the graft (FIG. 4o,p ).

As in mice (FIG. 13), serotonergic and GABAergic cells were rare (<1% oftotal cells) in rat cells, as were the mostly host-derived GFAP+ glialcells (7% of total cells; (FIG. 14). While few serotonin+ neurons weredetected in the graft, hNCAM-negative cells were observed that werelikely host-derived serotonergic fibers (FIG. 14).

Example V

Engraftment of novel DA neuronal cell population in primates containingdamaged neurons.

The results demonstrated herein showed excellent graft survival andbehavioral outcome in two independent murine models. However, the numberof DA neurons required in a mouse or rat brain represents a smallfraction of the larger number of cells needed for engrafting in primatesand humans. To test the scalability of this protocol, performed pilotgrafting studies were done in two adult MPTP lesioned rhesus monkeys.

Batches of 50×10⁶ transplantable DA neuron precursors were obtained byday 25 of differentiation using the floor plate-based protocol. Classicdose for inducing a Parkinson-like condition was though a 3 mg MPTP-HCLinjected into the carotid artery (range 0.5-5 mg). This was followed bysystemic injection of MPTP at 0.2 mg/kg IV of MPTP. Cells were injectedat three locations (posterior caudate and pre-commissural putamen) oneach side of the brain (6 tracts in total, 1.25×10⁶ cells/tract), andthe animals were immunosuppressed with cyclosporine-A. One side of thebrain was injected with DA precursors from a GFP expressing subclone ofH9, while the other side was engrafted with cells derived from unmarkedH9 cells. Results showing engraftment of neurons in rhesus monkeys withcontinued FOX2A expression and TH production are shown in FIG. 4q-t .One month after transplantation, robust survival of midbrain DA neuronswas observed based on expression of GFP (FIG. 15) and the human specificcytoplasmic marker (SC-121) (FIG. 4q ). Each graft core was surroundedby a halo of TH+ fibers extending up to 3 mm into the host (FIG. 4r ).The graft cores were composed of TH+ neurons co-expressing SC-121 (FIG.4s ) and FOXA2 (FIG. 4t ). SC-121 and GFP negative areas within thegraft contained Iba1+ host microglia (FIG. 15) indicating incompleteimmunosuppression. In summary, engraftment of novel DA neuronal cellpopulation in primates, i.e. adult MPTP (3 mg of of MPTP-HCL(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ranging in concentrationfrom 0.5-5 mg MPTP-HCl) lesioned rhesus monkeys containing a severe >95%loss of endogenous midbrain DA neurons. MPTP exposure caused observablechanges and symptoms similar to Parkinson's disease in humans.

Example VI

Comparable differentiation potential towards midbrain DA neuron fate ofPINK1 mutant PD-iPSC cells versus wild-type hES (or iPSC) cells.

This example described the discovery that large populations of midbrainDA neurons developed with characteristics of a PD patient's neurons whena PD patient's cell line, i.e. PINK1 mutant PD-iPSC cell, obtained in amanner that did not result in the destruction of an embryo, were used asthe cell population for obtaining FOXA2/LIM1XA/TH+DA neurons of thepresent inventions.

PINK1 Q456X mutant PD-iPSC line was differentiated using the novelfloor-plate based midbrain DA neuron protocol (method) of the presentinventions which yielded midbrain differentiation profiles comparable tothose obtained from the iPSC H9 line. (FIG. 20). A-C) Immunocytochemicalanalysis of PINK1 mutant PD-iPSC line at day 11 of differentiation(midbrain precursor stage) for FOXA2 (red), LMX1A (green) and DAPI(blue) (A), day 25 of differentiation (early postmitotic DA neuronalstage) for FOXA2 (red) and TH (green) (B) and for NURR1 (red) and TH(green) (C). D-F) Same set of immunocytochemical analyses performedusing H9 derived cells at day 11 of differentiation for FOXA2 (red),LMX1A (green) and DAPI (blue) (D), at day 25 of differentiation forFOXA2 (red) and TH (green) (E) and for NURR1 (red) and TH (green) (F).

PINK1 mutant PD-iPSC showed PD like phenotype of protein aggregationfollowing long-term differentiation and maturation in vitro. Theinventors discovered that PINK1 mutant PD-iPSC showed evidence ofα-synuclein (major component of Lewy body on PD patience) expression incytosol of TH+DA neurons at day 55 of differentiation using the novelfloor-plate based midbrain DA neuron induction protocol, (FIG. 21a-b ).A, B) Immunocytochemical analysis of PINK1 mutant PD-iPSC line at day 55of differentiation for α-synuclein (LB509, red), TH (green) and mergedimage (A) and α-synuclein (red) and ubiquitin (green) (B). Theseα-synuclein positive cells also showed high expression of ubiquitin(classical Lewy body marker). In contrast, DA neurons derived fromcontrol iPS line showed expression of normal synaptic (as opposed tocytosolic) α-synuclein expression and very low levels of Ubiquitin (FIG.21c-d ). C, D) Immunocytochemical analysis of control-iPSC line at day55 of differentiation for α-synuclein (red) and TH (green) (C) andα-synuclein (red) and ubiquitin (green) (D).

Expression of aggregated form of α-synuclein. In the PD patient brain,dimerized insoluble forms of α-synulcein lead to aggregation in Lewybodies. The dimerized form of α-synuclein shows phospholylation ofSerine 129 on α-synuclein.

At the same day of differentiation, PINK1 mutant PD-iPSC derived cellsshowed strong expression for Ser129 phosphorylated α-synuclein incontrast to control-iPSC derived cells that showed very low levels ofexpression (FIG. 22). PINK1 mutant PD-iPSC derived cells showed strongexpression for Ser129 phosphorylated α-synuclein in contrast tocontrol-iPSC derived cells that showed very low levels of expression. A,B) Immunocytochemical analysis for Ser129 phosphorylated α-synuclein(green) and DAPI (blue) in PINK1 mutant PD-iPSC derived cells at day 55of differentiation (A) and matched control-iPSC derived cells (B).

Differences in α-synuclein expression patterns are observed depending ofdifferentiation protocol. The inventors contemplated that floor-platederived “authentic” midbrain DA neurons showed PD specific vulnerabilityand corresponding, specific, in vitro phenotypes. DA neurons obtainedusing the classical MS5 stromal feeder based differentiation protocol(Perrier et al., PNAS 2004, herein incorporated by reference) yieldedlarge numbers of TH+ neurons. However, based on data obtained during thedevelopment of the present inventions, the inventors showed that MS5based TH+ cells were not authentic floorplate derived midbrain DAneurons. In cultures differentiated via the MS5 protocol, there weremany α-synuclein positive cells. However, those cells did not co-expressTH. Moreover, there was no difference in expression patterns betweenPD-iPSC and control-iPSC when using the MS5 differentiation strategy(FIG. 23a-b ). These data indicate that α-synuclein is also expressed inother non-DA cell types and that such non-DA α-synuclein is unchanged indisease versus control-iPSC derived cells—particularly when usingstandard MS5 differentiation protocols. These are the DA-like rosettederived neurons reported in publications (e.g. Perrier PNAS 2004). ThoseMS5 based TH+(=DA-like) cells are used for comparison in FIGS. 3, 10, 13and 16. These data indicate that α-synuclein is also expressed in othernon-DA cell types and that such non-DA α-synuclein is unchanged indisease versus control-iPSC derived cells, particularly when usingstandard MS5 differentiation protocols. Finally, the new floor platebased differentiation protocol described herein, yields large number ofTH+ cells co-expressing α-synuclein. Those TH+ cells express α-synucleinin a cytosolic expression pattern. FIG. 24A, B) Immunocytochemicalanalysis for α-synuelein (LB509, red), TH (green) of PINK1 mutantPD-iPSC line at day 60 of MS5 based differentiation (A) and control-iPSC(B). C) Immunocytochemical analysis of PINK1 mutant PD-iPSC line at day55 of floor-plate based differentiation for α-synuclein (red), TH(green).

Exemplary DA neurons derived from PINK1 mutant PD-iPSC are morevulnerable to toxic stimulation. PD-iPSC derived TH+DA neurons derivedvia floor-plate based protocol were more vulnerable to toxin challenge(valinomycin: mitochondria ionophore, 5 uM (ranging in concentrationfrom 1-10 uM), 48 hr) than control-iPSC derived cells. In contrast, TH+neurons derived via the classic MS5 based protocol did not showdifferential vulnerability between PD-versus control-derived cells.(FIG. 24). A-F) Representative TH immunocytochemistry at day 60 ofdifferentiation: Normal condition (no toxin treatment) for both PD- andcontrol-iPSC derived cells obtained via floor-plate based protocol (A,PD-iPSC derived cells shown), nearly complete degeneration of TH+DAneurons in PD-iPSC following toxin treatment (B), partially degeneratedTH+DA neurons from control-iPSC (C). Entire cell viability assay withalamar-blue after 48 hrs of valinomycin treatment also showeddifferential cell survival in a specific dose range for toxin challenge(5 and 10 uM) when comparing PD-iPSC and control iPSC (FIG. 25).

Normal condition both of PD- and control-iPSC derived cultures obtainedvia MS5 based protocol (D, PD-iPSC derived cells shown), TH+ neuronsfollowing toxin challenge in PD-iPSC (E), and control-iPSC derivedcultures (F) obtained via MS5 protocol. G-H) low power images ofimmunocytochemistry for Tuj 1 (red) and TH (green) by floor-plate basedprotocol at day 60 of differentiation: PD-iPSC of normal (G), versustoxin challenge (H) conditions and control iPSC of normal (I), versustoxin challenge (J) conditions. K-N) low power images ofimmunocytochemistry for Tuj 1 (red) and TH (green) by MS5 based protocolat day 60 of differentiation: PD-iPSC of normal (K), versus toxinchallenge (L) conditions and control iPSC of normal (M), versus toxinchallenge (N) conditions.

Exemplary quantification of cell viability-dose response assay for toxinchallenge. Cell viability assay with alamar-blue after 48 hrs ofvalinomycin treatment showed differential cell survival in a specificdose range for toxin challenge (5 and 10 uM) when comparing PD-iPSC andcontrol iPSC (day 60 of floor-plate based differentiation). Note: thisassay tests for overall cell death while the most dramatic effects wereobserved specifically in DA neurons (see FIG. 14). Therefore, alamarblue based quantification will likely underestimate the extent of thedifferential effect observed on DA neuron lineages.

References, herein incorporated by reference: Li, et al. Nat.Biotechnol. 23, 215-221 (2005); Perrier, et al. Proc Nati Acad Sci US101, 12543-8 (2004); Perrier, et al. Proc Natl Acad Sci USA 101, 12543-8(2004); Tabar, et al. Nature Med. 14, 379-381 (2008); Perrier, et al.Proc Natl Acad Sci USA 101, 12543-8 (2004); Wernig, et al. Proc. Natl.Acad. Sci. U S. A 105, 5856-5861 (2008); Lindvall, et al. J. Clin.Invest 120, 29-40 (2010); Roy, et al. Nature Med. 12, 1259-1268 (2006);Elkabetz, et al. Genes Dev. 22, 152-165 (2008); Kittappa, et al. PLoS.Biol. 5, e325 (2007; Ferri, et al. Development 134, 2761-2769 (2007);Roelink, et al. Cell 76, 761-775 (1994); Liem, et al. Cell 82, 969-979(1995); Fasano, et al. Cell Stem Cell 6, 336-347 (2010); Chambers, etal. Nat. Biotechnol. 27, 275-280 (2009); Muroyama, et al. Genes Dev. 16,548-553 (2002); Joksimovic et al. Nat Neurosci 12, 125-131 (2009);Lyashenko, et al. Nat. Cell Biol. 13, 753-761 (2011); VanDunk, et al. J.Neurosci. 31, 6457-6467 (2011); Huang, et al. Nat. Protoc. 4, 44-57(2009); Costa, et al. Mol. Cell Biol. 9, 1415-1425 (1989); Elkabetz, etal. Genes Dev. 22, 152-165 (2008); Soldner, et al. Cell 136, 964-977(2009); Guzman, et al. J. Neurosci. 29, 11011-11019 (2009); Nedergaard,et al. J. Physiol 466, 727-747 (1993); Ferrari, et al. Eur. J. Neurosci.24, 1885-1896 (2006); Olanow, et al. Trends Neurosci. 19, 102-109(1996); Zetterstrom, et al. Science 276, 248-250 (1997); Quintana, etal. Nature 456, 593-598 (2008); Kim, et al. dicts Neural DifferentiationPropensity in Human Pluripotent Stem Cells. Cell Stem Cell 8, 695-706(2011); Hargu, et al. Proceedings of the National Academy of Sciences ofthe United States of America 107, 15921-15926 (2010); Aubry, et al.Proc. Natl. Acad. Sci. USA 105, 16707-16712 (2008); Blume, et al., Exp.Neurol. 219, 208-211 (2009); Ban, et al., Proc. Natl. Acad. Sci. USA(2011); Studer, et al., Nature Neurosci. 1, 290-295 (1998); Kordower, etal., Science 290, 767-773 (2000); Paxinos, et al., The Rhesus MonkeyBrain in Stereotaxic Coordinates(Academic Press, 2000); Crawley, What'sWrong With My Mouse: Behavioral Phenotyping of Transgenic and KnockoutMice (Wiley-Liss, 2000); Studer, et al., Brain Res. Bull. 41, 143-150(1996); Tabar, et al., Nat. Biotechnol. 23, 601-606 (2005); andPlacantonakis, et al., Stem Cells 27, 521-532 (2009).

Example VII

Exemplary conditions were established for the in vivo recording of humanpluripotent stem cell derived DA neurons in acute slice preparations;see exemplary results shown in FIG. 26.

Electrophysiological measurements are contemplated for use in acuteslice preparations, i.e. from biopsies of engrafted areas. In oneembodiment, A9-versus A10 type graft-derived DA neurons will beidentified in vivo based on testing for the autonomouse pacemakingactivity that is specific to A9-type dopamine neurons that are mostaffected in PD. In other words, A10 type neurons do not have pademakingactivity

Conditions were established for the in vivo recording of humanpluripotent stem cell derived DA neurons in acute slice preparations,see, FIG. 26. Specifically, grafted human DA neurons derived frompluripotent stem cells were measured for and discovered to haveelectrophysiological features typical of those seen in mouse substantianigra pars compacta (SNpc), FIG. 26A hwere the top view showsreconstruction of a pacemaking neuron in the graft region. Bottom showsan exemplary photomicrograph of a brain slice taken from the rat intowhich the hES-derived neurons were injected 9 months prior; the graft isoutlined; a higher magnification image is shown inset at the bottom. Theslice was processed for tyrosine hydroxylase which shows up as white,FIG. 26B. Further, the top view shows an exemplary cell-attached patchrecording from a putative DA neuron in the graft; Bottom shows anexemplary whole cell recording from the same cell. Recordings were madein the presence of glutamate and GABA receptor antagonists (50 μM AP5,10 μM CNQX and 10 μM GABAzine) to eliminate synaptic input. Theserecordings demonstrated that the PS-derived neurons were autonomouspacemakers with normal intrasomatic voltage trajectories. Another neuronrecorded in a graft sample had similar properties, FIG. 26C. Forcomparison, cell-attached and whole cell recordings from a dopaminergicneuron in SNpc of an adult mouse are shown. Abbreviations (CTx=cortex,STr=striatum, SNpc=substantia nigra pars compacta, DA=dopaminergic).This data shows in vivo functional studies in grafted rat striatummonths after transplantation. Thus in some embodiments, in vivofunctional studies on grafted tissue demonstrates recovery of substantianigra pars compacta (SNpc).

Example VIII

Exemplary methods for identifying cell surface markers for use inmethods of the present inventions. In particular, CD142 was identifiedwith these methods.

Two main strategies to identify candidate surface markers: An unbiasedgene expression screen in genetic reporter lines (FIG. 27a ) that foundseveral candidate markers, including a marker, termed DCSM1, that isselectively expressed in midbrain DA neurons and appears to speciallymarker A9-type DA neurons (FIG. 27b ). A second strategy is the use of aCD cell surface marker screen in hESC derived DA neurons testing 242commercially available antibodies in 96 well format (FIG. 27c,d ). Theresults of such a screen (FIG. 27e ) led to the identification of atleast 5 validated markers enriched in midbrain DA neurons includingCD142, a marker that selectively marks Nurr1+DA neuron stage (FIG. 27f). With the use of the DA neron cell procedure described herein, CD142typically marked approximately 30% of the total cell population at day25 of differentiation (FIG. 28a ). Selectivity of CD142 for a Nurr1+ DAneuron stage was confirmed in multiple independent hESC and hiPSC lines(FIG. 28b ). In addition to enriching for DA neurons, enrichment ofCD142 positive cells resulted in selective depletion of undesired neuronsubtypes such as GABA and Serotonergic neurons. (FIG. 28c-f ). in vivostudies confirmed the ability of a CD142 positive cell population togive rise to high purity DA neuron grafts that overcame problems ofcontaminating GABA and Serotonergic neurons. While the graftingprocedure that used unpurified cells already resulted in very fewSerotonergic neurons, the use of CD142 based selection of precursorcells is contemplated to further reduce the risk of introducingserotonergic neurons, a contaminating cell type that was implicated infailed human fetal tissue grafting as the potential source of theundesirable fetal tissue graft-induced dyskinesias.

Example IX

This example describes methods for transformation of cells with humanPST genes for increase PSA cell surface expression. This example alsoshows exemplary methods of using cells having increased PSA cell surfaceexpression.

Specifically, this example shows engineered PST genes into hESCs forincreasing PSA expression on DA neurons. A gene encoding the humanpolysialyl-transferase (hP ST) was introduced into a hESC line (VVA01)using a lentiviral vector (pLenty, Invitrogen). Twenty selected cloneswere expanded and analyzed for PST expression.

PST-expressing hESC clones were differentiated to ensure that PST wasnot silenced in DA neurons. Quantification of PSA-NCAM at differentstages of differentiation (day 0, 11, 25, and 50) was done using FACSanalysis and immunofluorescence (Operetta). Positive clones weresubjected to the suite of DA neuron QC parameters outlined in Table 7.At least 3 clones that retain high, uniform levels of PSA-NCAM duringdifferentiation and perform well in the QC parameters (Table 7) willadvance to assessment of the neurite outgrowth in PST-overexpressinghESC-derived DA neurons Selected control and PST-overexpressing hESCclones were differentiated into DA neurons using the standard protocoldescribed herein, followed by cell fixation and analysis at days 25 and50. The number and length of TH-positive fibers in such cultures werequantified with the Operetta High Content Microscope. The NeuriteAnalysis module in Harmony software 3.0 quantified neurite number andlength, with or without PST, and the data was statistically analyzedusing a two-way ANOVA. PST-overexpressing and control hESC clones thatadvance from in vitro studies above, were differentiated again into DAneurons and transplanted into a rat model of PD. Short-term grafts (4-6weeks) to determine survival, PSA-NCAM expression and neurite outgrowthwere done. For each clone that passed short-term in vivo parameters weresubjected to long-term grafting studies. For those studies animalsreceived half or a quarter of the standard (200×10³) dose of cells.These studies were to address whether increased PSA leads to increasedlong-term survival after transplantation (5 months), and whether smallerDA neuron numbers are capable of matching or outperforming thefunctional capacity of non-PST grafts transplanted at standard celldoses (not FIG. 27). In addition, complex behavioral assays sensitive tothe extent of striatal reinnervation were monitored to furtherdistinguish the functional potential of PST-versus control DA neurongrafts. The animals were sacrificed following completion of behavioralassays, and fiber outgrowth was quantitated using human specificantibodies NCAM and SC121 and antibodies against TH (see also not FIG.29). The intensity and spread of the hNCAM+, SC121+ and TH+ graft wasmeasured, as well as the percentage of human cells co-expressing DAneuron markers (TH, FOXA2) and PSA. The density of NCAM/TH+ halo ofneurites emanating from the graft were quantified at differentdistances. Data was compared among groups using a two-way ANOVA with aBonferroni post-hoc test. In addition, sections were examined forqualitative changes (e.g. branching, thickness, graft distribution andshape). In addition, some grafts will be processed for sliceelectrophysiological evaluation in terms of A9 phenotype, synapseformation with host striatum, as well as innervation by endogenousafferents.

Example X

The following example shows enhancement of polysialic acid expressionthat improved the function of ES-derived dopamine neuron grafts inParkinsonian mice.

ES cells expressing GFP under control of Nurr1 promoter (Nurr1::GFP EScells) were stably transduced with a lentiviral vector ubiquitouslyexpressing polysialyltransferase (PST). Transduced cells showed adramatic increase in PST mRNA as compared to controls (FIG. 30A).Expression of PST was observed to be sufficient for PSA synthesis onNCAM. Accordingly, PSA-NCAM expression was greatly increased inPST-modified cells at day 14 of DA neuron differentiation (FIG. 30B-E).Both the endogenous and induced cell surface PSA on ES-derived DAneurons could be removed (FIG. 30E) by a phage endoneuraminidase (endoN)that specifically cleaved PSA's unique alpha-2,8-linked sialic acidpolymers. Surprisingly, PST transduction was not observed to affectexpression of neuronal or midbrain markers in the GFP-purified DAneurons (FIG. 30F).

Other studies in 6OHDA-lesioned hemiparkinsonian mice showed thattransplantation of approximately 100,000 ES-derived DA neuron precursorsis required to produce robust functional recovery, as measured by theamphetamine-enhanced rotation test. In the present studies, sought tograft a sub-optimal number of cells in order to be able to assessaugmentation by enhanced PSA expression. In order to transplant highlyenriched DA neuron populations that are depleted for contaminatingpluripotent cells, FACS-purified cultures at day 14 of differentiationfor expression of Nurr1-driven GFP and for the absence of SSEA-1expression (FIG. 31). Without PST overexpression, a reduction of theminimally effective graft size by half (55,000 Nurr1+DA cells) failed toproduce detectable behavioral recovery. By contrast, with enhanced PSAexpression, the same number of Nurr1/PST DA neurons resulted in asignificant correction of the PD behavioral impairment (p<0.01; two-wayANOVA), with complete recovery approximately 5 weeks after surgery (FIG.32A). PSA removal prior to transplantation by incubation with endoNindicated the specificity of PSA's enhancement, in that the endoNtreatment partially reversed the functional restitution obtained withNurr1/PST (FIG. 32A).

To examine the characteristics of the grafted cells, animals wereprocessed for immunohistochemistry two months after transplantation.There was a difference in the number of surviving Nurr1+ neurons, inthat animals grafted with the PST-transduced line had on average twiceas many GFP+ cells as animals grafted with control cells (9,300+/−1,400vs. 4,230+/−1010 GFP+ cells per graft in PST versus control samplesrespectively; FIG. 32B, p<0.05, Student's t test). Furthermore,Nurr1/PST grafts also displayed higher levels of PSA expression in vivo(FIG. 32C,D). However, the proportions of cells expressing the midbrainDA markers TH and FoxA2 within the graft core were comparable for theNurr1 and Nurr1/PST cells (TH: 62.0%+/−8.0 vs. 51.3%+/−7.0 p=0.33;FoxA2: 63.2%+/−8.6 vs. 55.4%+/−2.0, p=0.3, respectively; FIG. 32E).

Neuronal processes that emerged from the Nurr1 and Nurr1/PST cellsshowed comparable levels of TH, Girk2 (G-protein-coupled, inwardlyrectifying potassium channel) and synapsin (FIG. 33A). Unlike otherstudies with transplanted Schwann cells (Ghosh, M., et al. Extensivecell migration, axon regeneration, and improved cells after spinal cordinjury. Glia 60, 979-992 (2012)), enhanced PSA expression had littleeffect on migration of DA cells from the grafting site. However, therewere clear changes in neurite outgrowth. As shown in FIG. 33B, therewere more DA neuronal processes emerging from Nurr1/PST cells ascompared to Nurr1+ controls. When the intensity of GFP and THimmunofluorescence was quantified in five successive 100 μm zones awayfrom the transplant, Nurr1/PST grafts displayed a much higher relativedensity of processes (FIG. 33C,D; p<0.01 for both GFP and TH, two-wayANOVA). In quantifying this effect, normalized the relative density ofprocesses to the density observed in the most proximal zone immediate tothe graft core. Such normalization was required to compensate for thelarger number of surviving cells in the Nurr1/PST grafts and to confirma specific effect of PSA on neurite outgrowth. Specificity was alsodemonstrated when cell surface PSA was removed by endoN treatment priorto grafting. Thus pre-treatment with endoN reduced distal fiberoutgrowth back to control levels (FIG. 33E).

These discoveries showed that at least some of the effects of PSA ongraft function resulted from enhanced fiber innervation of striatum.Accordingly, there was a strong correlation between graft function andthe relative extent of GFP-positive fiber outgrowth for example intozone IV (FIG. 33F; p<0.001, r2=0.65, n=17). Surprisingly, the fiberoutgrowth/behavioral relationship was consistent for experimental groups(control, PSA enhanced, and endoN-treated), indicating that graft-hostinnervation was a parameter for behavior recovery in the mouseParkinsonian model. Several factors contributed mechanistically toincreased fiber outgrowth, such as enhanced penetration of the zone ofreactive glia encapsulating the graft core, increased sprouting ability,improved outgrowth into the surrounding host tissue (e.g. easier growthcone translocation), and prevention of premature connections with hosttissue in proximity to the graft core. The exemplary mechanisms areconsistent with PSA's role in facilitating process outgrowth duringnormal development and in the adult nervous system.

The experiments described herein demonstrated the use of engineered PSAin DA neuron grafting which provided superior results compared to graftsfrom other types of cells. Data clearly indicated that PSA enhancementprovided a significant augmentation of the ability of grafted DA neuronsto innervate host striatum and attenuate PD functional deficits.Therefore clinical translation is contemplated comprising DA neurons ofthe present inventions for providing cells prior to transplantation. Insome embodiments, the cells will be genetically manipulated forexpressing PSA. In some embodiments, PST may be delivered directly tothe cells via exposure to the purified enzyme and substrate, in vitro,prior to transplantation. In some embodiments, PSA strategy for humantranslation in PD grafting is contemplated to minimize the need formultiple injections and thereby reduce the surgical risks resulting fromthese multiple injections.

In other embodiments, this technology is contemplated for use on othercell types and species, for example, augmenting the migration of graftedSchwann cells in creating a bridge (for example, cell-cellcommunication) for re-growth of axons at the site of spinal cord injury.

The following are exemplary materials and methods used in this example.

Animals: Six-week old 129S3/SvIrnJ mice (Jackson Laboratory) were keptunder controlled temperature with food and water available ad libitum.Experimental procedures were performed according to NIH andinstitutional animal use guidelines and approved by the localInstitutional Animal Care and Use Committee (IACUC) and theInstitutional Biosafety Committee (IBC).

6OHDA injection and amphetamine-induced test: Animals were anesthetizedwith sodium pentobarbital (10 mg/kg) and injected in the right striatumwith 2 μl of 6OHDA (4 μg/μl in saline, 0.5% ascorbic acid). Theinjections were performed with a Hamilton syringe at coordinates: 0.5 mmposterior, 1.8 mm lateral relative to bregma and 2.5 mm ventral to brainsurface. Before the surgery animals received a single i.p. injection ofdesipramine (25 mg/Kg, Sigma). Two weeks after surgery animals werescored in the amphetamine-induced rotation test. They were placed on 30cm diameter clear plastic cylinders for half an hour after which theyreceived a single i.p. injection of amphetamine (10 mg/Kg, Sigma). After20 min, the number of ipsilateral/contralateral rotations was scoredduring another 20 min. Animals were scored once a week for seven weeksthen deeply anesthetized and perfused through the heart with PBS and 4%paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains wereremoved and postfixed overnight at 4° C. in 4% paraformaldehyde thenvibratome sliced (Pelco-101, Ted Pella) in 40 μm-thick sagittalsections.

Cell differentiation and transplantation: A Nurr1::GFP BAC transgenicBAC mouse ES reporter cell line (i.e., GFP expression is driven by Nurr1promoter) 5 was transduced with a lentivirus (pLenti, Invitrogen)containing the mouse PST gene under control of the CMV promoter. EScells were propagated on mitomycin C-treated MEFs (StemCellTechnologies) in DMEM (Invitrogen), 10% FBS (HyClone) supplemented with1,400 units/ml LIF (ESGRO; Invitrogen), 2 mM L-glutamine, 1 mMI3-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin(Invitrogen). DA differentiation was induced according to Barberi etal., Nat Biotechnol 21, 1200-1207 (2003), with modifications. Briefly,cells were differentiated on MS5 feeder cells in gelatin-coated dishes(10,000 cells/10 cm dish) and cultured for four days on serumreplacement media (SRM). At day 4, Sonic hedgehog (SHH, 200 ng/ml) andFGF8 (100 ng/ml) were added. At day 7 of differentiation, the media waschanged to N2 supplemented with SHH, FGF8 and bFGF (10 ng/ml). At day11, terminal differentiation was induced by withdrawal of SHH, FGF8 andbFGF and the addition of ascorbic acid (AA, 200 μM) and BDNF (20 ng/ml).

Cells were harvested at day 14-15 with accutase treatment for 45 mM,washed once with N2 and incubated with AlexaFluor-647 conjugatedanti-SSEA-1 antibody (BD Pharmingen) for 25 min. Cells were washed oncewith N2, resuspended in HEPES buffer with 0.1% BSA. DAPI was added toassess viability. FACS was performed with a MoFlo cell sorter and thepopulation of interest was sorted for GFP fluorescence (Nurr1). Thepopulation positive for AlexaFluor-647 (SSEA-1) was negatively sorted.For GFP negative control, naïve J1 mouse ES-cells were used at the samedifferentiation stage.

Nurr1::GFP sorted cells were analyzed for viability and resuspended inN2 with BDN and AA to a final concentration of 55,000 cells/μl. One μlwas injected into the lesioned mouse striatum with a 50 μm tipped fineglass capillary at coordinates: 0.3 mm posterior, 1.5 mm lateral frombregma and 2.2 mm ventral to the brain surface. An aliquot of the cellsuspension was re-plated in matrigel-coated 6 mm dishes for furthercharacterization.

For immunofluorescence analysis, cells were fixed with paraformaldehydefor 10 min at 4 0 C, washed twice with PBS, blocked with 5% BSA (0.1%Triton X-100 in PBS) and incubated with primary antibodies for 2 hrs atroom temperature: rabbit anti-GFP (1:1000, Invitrogen), mouse IgManti-PSA (1:2000, 5A5), mouse anti-NeuN (1:800, Chemicon), mouse anti-TH(1:1000, Sigma), goat anti-FoxA2 (1:800, Santa Cruz), goatanti-Engrailed (1:800, Santa Cruz). Cells were then incubated withCy-conjugated secondary antibodies (1:1000, Jackson).

EndoN treatment: To remove PSA from NCAM, the night before harvesting,cells were treated with 20 units of endoN, a phage enzyme thatspecifically removes PSA 7-9. Cells were then harvested and injected asdescribed before but were resuspended in N2 with BDNF and AA and 5 unitsof endoN. We previously assessed that the injection of the same amountof endoN alone into lesioned mice did not improve animal behavior.

PST mRNA and PSA-NCAM analysis in vitro: For Western blot analysis,cells were treated with WB buffer (PBS with 1% NP40, 150 mM NaCl, 1 mMEDTA, and 1× protease/phosphataseinhibitors added immediately beforeextraction, at pH of 7.4) and sonicated twice for 5 sec, centrifuged andresuspended in Laemli buffer (LB). Aliquots without LB were saved forprotein determination. Equal amounts of protein were loaded into 6%sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (BioRad).Proteins were transferred by electrophoresis onto polyvinylidenemembranes (Millipore). The membranes were blocked for 1-6 hr in 0.1%Triton X-100 TBS (TBS-T) with 5% non-fat dry milk and incubatedovernight with anti-NCAM antibody (1:10,000, Santa Cruz) in TBS-T with5% milk. Blots were then incubated with peroxidase-conjugated secondaryantibody (1:10,000, Jackson) and detected with ECL detection method(Amersham Pharmacia Biotech). Protein levels were quantified usingImageJ software.

For qRT-PCR analysis, total RNA was extracted with Trizol (Sigma),reverse-transcribed (Qiagen) and amplified with 10 μl of 2×SYBR reactionmixture and 0.2 μM of forward and reverse primers to a final volume of20 For PSA-NCAM FACS analysis, cells were harvested with accutasetreatment for 45 min, washed once and incubated with mouse IgM anti-PSA(1:250, 5A5) for 25 min on ice, washed once with N2 media and incubatedwith Cy3-conjugated anti-mouse-IgM (1:250, Jackson) for another 25 minon ice. Cells were washed once with N2 and resuspended with 0.1% BSAwith 7AAD and analyzed in a FACS Calibur cell sorter. As control, noprimary antibody was added.

Immunohistological and stereological procedures: Free floating coronalsections were blocked in 0.1% Triton X-100, 5% donkey serum in PBS for30 min at room temperature and incubated 48 hrs at 4° C. with differentantibodies: rabbit anti-GFP (1:300), chicken anti-GFP (1:200, Chemicon),mouse anti-TH (1:200), mouse IgM anti-PSA (1:1000), mouse anti-NeuN(1:400), goat anti-FoxA2 (1:300), rabbit anti-Girk2 (1:300, AlomoneLabs), mouse anti-synapsin (1:200, BD Transduction Laboratories).Sections were then washed and incubated with secondary antibodies: Cyt,Cy3 and Cy5-conjugated donkey antibodies (1:400, Jackson). For PSA aCy5-conjugated donkey anti-IgM was used (1:500 Jackson). Incubationswere performed for 2 hrs at room temperature. Sections were washed twicein PBS and mounted in Mowiol (Calbiochem). One-in-three coronal sectionsof the brain were analyzed for each immunolabeling. Digital images werecollected by a Zeiss LSM 510 laser scanning confocal microscope withthree lasers (Argon 488, HeNe 543 and HeNe 633) with a c-Apochromat40×objective (water-immersion). The number GFP+ and TH+ cells wascounted in one-in-three sections encompassing the whole brain under a40× objective, and the total number of cells/graft estimated.Double-labeled cells were analyzed in single optical planes through theentire z-axis.

For the analysis of the percentage of GFP/TH+ and GFP/FoxA2+ labeledcells, 100 GFP+ cells were analyzed for each marker. For processoutgrowth analysis, confocal z-scans were performed at 0.8 μm intervalsthrough the entire z-axis (20-40 μm) with a pinhole of 1 μm under a 40×objective. Sections were scanned from the injection site laterally untilno processes were observed. 3-D projections encompassing the wholescanned area were sequentially matched. For GFP and TH intensityanalysis, the entire scanned area was divided into five successive 100μm zones away from the transplant and the intensities were measuredusing ImageJ software. Data were normalized to the intensity in the zonenearest the graft (zone I) to control for any potential differences ingraft size.

Statistical analysis: Data are presented as the mean±standard error ofthe mean (SEM). Comparisons were performed using Student's t test ortwo-way analysis of variance (ANOVA) followed by Bonferroni post-hoctest. Linear regression analysis was performed and quantified using thePearson correlation.

Example XI

The following example shows enzymatic engineering of PSA on hESC-derivedDA neurons using the purified bacterial polysialyltransferase, PSTnm, toenhance transplant efficacy.

Although effective, PST gene transfection necessitated geneticmodifications of hESCs with limited control over the duration ofpolysialylation. This exemple describes the discovery that externalPSTnm induced PSA, instead of gene delivery, (see, FIG. 35). In FIG.35A, PST treated Schwann cells (SC) (green line-middle line) hadincreased adhesion time while PSTnm-produced PSA inhibited adhesion. Inparticular, (A) PSTnm-produced PSA inhibits adhesion of Schwann cells insuspension to a Schwann cell monolayer even more effectively (redline-lowest line) than PSA produced by forced PST expression (greenline-middle line). (B) PSA immunoblotting in ESC-derived HB9 motoneuronsshows that control samples treated with PSTnm alone had undetectablelevels of PSA. Incubation with PSTnm+ CMP-sialic acid substrate producesa large PSA band, which is removed with endoN treatment. (C, D) Similarto effects obtained with the PST gene, polysialylation of these cells byPSTrun and substrate during differentiation enhances neurite outgrowthand cell migration (arrowheads). (E) PSA immunostaining of day-30hESC-derived DA neurons. (F) This staining is significantly increasedafter treatment with PSTnm and substrate. (G) In vivo injection of PSTnm alone has no effect, while its co-administration with substrate (H)produces large amounts of PSA expression in mouse striatum.

Thus mature DA neurons externally treated with PSTnm is contemplated foruse in the producing cells for engraftment. Both mammalian PST and PSTnmproduced chemically identical chains of PSA. Increased PSA onhESC-derived DA neurons (FIG. 35F) should persist for several weeks,sufficient for DA fibers to exit graft core. Because PSTnm is removedprior to grafting, immunogenicity to this enzyme contaminating thegrafted cells should not be factor.

PSTnm was produced from an engineered fragment with enhanced solubilityand activity characteristics (Willis et al., Characterization of thealpha-2,8-polysialyltransferase from Neisseria meningitidis withsynthetic acceptors, and the development of a self-primingpolysialyltransferase fusion enzyme. Glycobiology 18, 177-186 (2008)).Cultures of hESC were induced to differentiate into DA neurons beforePSTnm exposure, exposure to substrate or both. Cultures were examined atdifferent time-points of exposure (10 min to 6 hrs) by quantitativeimmunofluorescence (Operetta) and western blotting to determine thespeed and levels of polysialylation. Thus, Day 25 differentiatedhESC-derived DA neurons will be incubated with the optimumconcentrations of PSTnm and substrate using the conditions describedherein. PSA+ mDA neurons will be transplanted in short- and long-termassays as described herein and in FIG. 29.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention was described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in cellularbiology, neurobiology, cancer cell biology, molecular biology,biochemistry, chemistry, organic synthesis, or related fields areintended to be within the scope of the following claims.

What is claimed is:
 1. An in vitro method for differentiatingpluripotent stem cells, comprising exposing a plurality of pluripotentstem cells to at least one inhibitor of Small Mothers AgainstDecapentaplegic (SMAD) signaling, and exposing the cells to at least oneactivator of Sonic hedgehog (SHH) signaling and at least one activatorof wingless (Wnt) signaling, wherein the cells are exposed to the atleast one activator of Wnt signaling three (3) days from the initialexposure of the cells to the at least one inhibitor of SMAD signaling toobtain a cell population comprising at least about 10% differentiatedcells expressing both forkhead box protein A2 (FOXA2) and LIM homeoboxtranscription factor 1 alpha (LMX1A).
 2. The method of claim 1, furthercomprising subjecting the cell population to conditions favoringmaturation of the differentiated cells into dopamine neurons.
 3. Themethod of claim 2, wherein the conditions comprise exposing the cellpopulation to at least one of brain-derived neurotrophic factor (BDNF),ascorbic acid (AA), glial cell line-derived neurotrophic factor (GDNF),dibutyryl cAMP (dbcAMP), and transforming growth factor type β3 (TGFβ3).4. The method of claim 2, wherein the dopamine neurons express at leastone marker selected from the group consisting of (a) tyrosinehydroxylase (TH), orthodenticle homeobox 2 (OTX2), nuclear receptorrelated 1 protein (NURR1), neuron-specific class III beta-tubulin(Tuj1), Trefoil factor family 3 (TTF3), paired-like homeodomain 3(PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1),early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopaminetransporter (DAT), and G-protein coupled, inwardly rectifying potassiumchannel (Kir3.2/GIRK2), CD142, DCSM1, CD63, CD99, and ALDH1; or (b)Girk2, CD142, DCSM1, CD63, CD99, and ALDH1.
 5. The method of claim 4,wherein the dopamine neurons are A9 subtype neurons.
 6. The method ofclaim 4, further comprising selecting a population of cells expressingat least one marker selected from the group consisting of Girk2, CD142,DCSM1, CD63, CD99, and ALDH1.
 7. The method of claim 2, wherein the stemcells are differentiated into the dopamine neurons no later than about25 days from the initial exposure of the cells to the at least oneinhibitor of SMAD signaling.
 8. The method of claim 1, wherein thepluripotent stem cells are selected from the group consisting ofembryonic stem cells, induced pluripotent stem cells (iPSCs), andengineered stem cells.
 9. A cell population comprising in vitrodifferentiated cells derived from stem cells according to the method ofclaim
 1. 10. A cell population comprising in vitro differentiated cells,wherein at least about 10% of the differentiated cells express FOXA2 andLMX1A.
 11. A cell population comprising at least about 10% cellsexpressing at least one marker selected from the group consisting ofGirk2, CD142, DCSM1, CD63, CD99, and ALDH1.
 12. The cell population ofclaim 11, comprising at least 20%, at least about 30% or at least about80% of cells expressing the at least one marker.
 13. The cell populationof claim 11, wherein the cells expressing the at least one markerfurther express a second marker selected from the group consisting ofFOXA2, LMX1A tyrosine hydroxylase (TH), nuclear receptor related 1protein (NURR1), neuron-specific class III beta-tubulin (Tuj 1), Trefoilfactor family 3 (TTF3), paired-like homeodomain 3 (PITX3), achaete-scutecomplex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3(EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), andG-protein coupled, inwardly rectifying potassium channel (Kir3.2/GIRK2).14. The cell population of claim 13, wherein the cells expressing the atleast one marker further express tyrosine hydroxylase (TH), and/orNURR1.
 15. The cell population of claim 11, wherein the cells expressingthe at least one marker are midbrain dopamine neurons.
 16. The cellpopulation of claim 11, wherein the cells expressing the at least onemarker are A9 subtype neurons.
 17. The cell population of claim 11,wherein the cells are derived from stem cells according to the method ofclaim
 1. 18. A pharmaceutical composition comprising the cell populationof claim 11 and a pharmaceutically acceptable carrier.
 19. A method oftreating at least one symptom in a subject having a neurologicaldisorder characterized by reduction of midbrain dopamine neuronfunction, comprising administering to the subject the cell population ofclaim
 11. 20. A method for isolating midbrain dopamine neurons andprecursors thereof from a population of cells, comprising isolatingcells that express a detectable level of at least one marker selectedfrom the group consisting of Girk2, CD142, DCSM1, CD63, CD99, and ALDH1.